Drugmonoclonal Antibody Conjugates For Targeting To The Lung

Another drug-carrier system that has been investigated as a means of achieving specific tissue targeting is the use of an antibody directed against the tissue that is the proposed site of action of the drug. Although this approach is not new, because pioneering work in this area was carried out as early as 1958 [202]. Recently, it has become evident that monoclonal antibody (mAb) biotechnology is effective in a wide range of disease. The current estimated market for these agents is about $25 billion each year. The application of antibodies is broad ranging and includes therapeutics, diagnostic tools, and research tools. The first therapeutic antibodies were mouse monoclonal antibodies that were selected against cytokines and cell surface proteins of proinflammatory, immunologic, or cancer cells [203]. The development of monoclonal antibodies to human tumor-associated antigens has been achieved, and this has led to a renewed interest in the use of drug-antibody conjugates for cancer therapy [204,205].

There are several questions that need to be addressed before considering the use of drug-monoclonal antibody conjugates.

1. Are sufficiently lung-specific antibodies available?

2. Is there evidence that such antibodies will localize only in lung tissues and not in other tissues in vivo?

3. Do the antibodies contain appropriate functionalities to enable covalent linkage of drug molecules, and, if so, will the conjugated antibody exhibit targeting characteristics similar to those of the parent antibody?

4. Will the formation of an antibody-drug conjugate result in an immunologically active entity on repeated administration?

With regard to these considerations, it is important to determine whether, after either regional or systemic administration, a drug-antibody conjugate can deliver potentially therapeutic doses of the drug. This may depend on the loading of drug at multiple sites of conjugation on the antibody surface. In this respect, the greater the number of drug molecules conjugated to each antibody molecule, the more ineffective the resultant molecule might be, because attachment of drug molecules near or around the antibody-antigen binding site or at locations that compromise the three-dimensional integrity of the antibody will lead to decreased tissue specificity or increased immunological activity, respectively. Thus, an evaluation of the therapeutic index of the conjugate must be undertaken to determine whether it is superior to the free drug.

When considering the targeting of therapeutic agents to lung tissue by this approach, the choice of cell surface antigens would appear to be most appropriate, although there is evidence that antibodies that can recognize in vivo antigens that are expressed extracellularly [206] can also be used for specific tissue targeting. A recombinant humanized mAb to human IgE has been found to inhibit mast-cell-dependent airway narrowing and other components of the asthmatic inflammatory response [207]. Currently, omalizumab is undergoing clinical testing for a number of indications, including asthma. Omalizumab is a mAb that specifically recognizes human IgE [208].

The most commonly used route of administration of antibody-drug conjugates has been the intravenous route, but intracavity administration has also been investigated; and, generally, localization of antibody conjugates in the targeted tissue by this latter route appears to be superior to the intravenous route [209]. The mechanism of antibody-drug delivery at the cellular level is believed to involve initial binding to the specific cell surface antigen. This binding is followed by internalization and endocytosis into lysosomes, where digestion of the conjugate by lysosomal proteinases would release free drug from where it would diffuse to the site of action [210].

As mentioned previously, a significant reduction in antibody reactivity is often observed after multiple sites of conjugation of drug with antibody. This has led to the development of carriers that, when covalently linked to antibody, are able in turn to covalently bind many drug molecules to appropriate carrier functionalities. Examples of carrier molecules that have been used include dextran [211-213], human serum albumin [214], and poly-L-glutamate [215]. Obviously, such a gross molecular modification of the parent antibody may well affect its overall properties, and this often leads to significant differences in biodistribution of an antibody-carrier-drug complex relative to the parent antibody. In addition, for reasons stated previously, such a structural derivitiza-tion may also result in lower tissue specificity and increased toxicity.

More innovative approaches to the targeting of drugs by antibody-drug complexes involve the use of hybrid-hybrid antibodies [216-218]. This approach uses hybrid antibodies with dual specificity, one site binding with, say, a cell surface antigen and the other with drug or cytotoxic agent. Such bispecific antibodies are prepared by reassociation of enzyme-prepared fragments of two antibodies or by hybridization of two existing hybridomas, one producing antibody to cell antigen and the other producing antibody to drug. The application of this strategy to the development of therapeutic agents could be twofold: Bispecific antibody would be initially administered, resulting in specific tissue localization (i.e., pretargeting), followed by drug, some of which would be taken up by the localized antibody; alternatively, binary complexes of both drug and antibody could be preformed in vitro and then administered, or drug and antibody could be given simultaneously. The concept of pretargeting is not new and has been investigated for diagnostic tumor imaging with the high-affinity avidin-biotin system [219]. In this system, antibody conjugated to avidin was evaluated for localization in tumors followed by administration of radiolabeled biotin. It is conceivable that such a system may be useful for the pretargeting of drugs to the lung, by using a biotin-drug conjugate in place of biotin.

The use of drug-antibody fragments has been examined as a means for tissue targeting [220,221]. Generally, fragments appear to be poorer targeting agents than intact antibody, although they do exhibit relatively faster overall clearance and catabolism, which may result in less systemic toxicity. However, studies indicate that the increased clearance and kidney metabolism of antibody fragments may lead to renal toxicity [222]. The use of antibody fragments lacking a more immunogenic portion of the intact antibody molecule has generally not resulted in a decrease in immunogenic properties.

Some recent reports involving monoclonal antibodies directed against epithelial and endothelial cell surface components suggest that this approach to drug targeting in the lung may have good potential. For example, monoclonal antibodies to a glycoprotein involved in fibronectin-mediated adhesion mechanisms has been reported for fibroblasts [223], and the injection of rabbit antisera or purified antibodies against basement membrane proteins type IV collagen and laminin into inbred mice results in cellular infiltration of mainly lung and kidney tissue. Antibody location was shown to be on the basement membranes of glomeruli, alveoli (pronounced), choroids plexus, liver, and blood vessels and in epidermal junctions [224].

Developments in the cancer area are also worthy of mention. An immunotoxin consisting of a murine monoclonal antibody (B4G7) that recognizes EGF receptor conjugated with gelonin, a ribose-inactivating protein, was specifically cytotoxic to EGF receptor-hyperproducing cells in mice and was nontoxic at 250 mm conjugate per mouse [225]. The results suggest that this conjugate may be useful for target therapy to epidermal growth factor receptor-hyperproducing squamous carcinoma. Also, a 125I-labeled monoclonal antibody directed against MW 48,000 human lung cancer-associated antigen may be useful in the diagnosis and treatment of lung cancer [226]. Chan et al. [227] reported that a set of mouse monoclonal antibodies directed against the c-myc oncogene product, a 62,000-d nuclear binding protein involved in cell cycle control, was constructed by immunization with synthetic peptide fragments. After intravenous administration, these monoclonal antibodies exhibited good tumor localization with primary bronchial carcinoma patients, thus indicating that monoclonal antibodies directed against oncogene products may provide novel selective tools for diagnosis and targeted therapy of cancer. Utilization of mAb to prevent cancer cell metastasis has been examined too. Studies have been done to utilize mAb such as the inhibitor of lung endothelial cell adhesion molecule (anti-lu-ECAM-1) to inhibit colonization of the lung by lung metastatic murine B16 melanoma cells [228]. Lung endothelial cell adhesion molecule (Lu-ECAM-1) has been isolated and characterized [229]; this molecule selectively binds lung-metastatic melanoma cells [230]. In a similar manner mAb 6A3 selectively binds a membrane glycoprotein of rat lung capillary endothelia and has been shown to inhibit specific adhesion of lung endothelial vesicles to lung metastatic breast cancer cells.

Because most monoclonal antibodies that have been studied for tissue targeting are from mouse or, occasionally, from rat, the problem of antibody production to such foreign proteins always exists. While murine-derived mAbs are well tolerated for acute therapy, their use in chronic therapy is limited, due to severe human anti-mouse antibody response (HAMA) [231]. The HAMA response is elicited due to the foreign nature of the antibody itself. Molecular engineering is being utilized to replace the foreign components of the murine antibody with human antibody sequences to overcome their immunogenicity [232].

Recently, successful attempts have been made to develop fully human monoclonal antibodies. Takacs et al. [102] mentioned in their review that, to date, greater than 12 fully human antibodies are currently being profiled in clinical studies.

Future directions for the more effective utilization of monoclonal antibodies as drug targeting agents must focus on a more rational design of the antibody-drug conjugate. Currently, attempts are under way to develop bispecific antibodies combining the VH and VL of two different antibodies into one molecule to ensure cellular targeting constitutes an effective disease treatment [233]. In particular, the chemistry of the linker groups in relation to release mechanisms at the site of action must be carefully evaluated. In addition, chemical entities that could be incorporated into the conjugate structure that may influence its biodistribution should also be investigated. Also, more emphasis should be placed on determining the precise mechanism of action to avoid misinterpretation of in vivo data. In instances where a therapeutic effect has been observed, little attempt has been made to determine whether site specificity has been achieved by the proposed mechanism. Finally, with the increasing availability of human monoclonal antibodies, it is clear that drug-antibody conjugates have even greater potential for clinical therapy, although the cost of manufacturing and purifying monoclonal antibodies still limits their clinical utility.

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