Tumor targeting using anti-her2 immunoliposomes

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Abstract

We have generated anti-HER2 (ErbB2) immunoliposomes (ILs), consisting of long circulating liposomes linked to anti-HER2 monoclonal antibody (MAb) fragments, to provide targeted drug delivery to HER2-overexpressing cells. Immunoliposomes were constructed using a modular strategy in which components were optimized for internalization and intracellular drug delivery. Parameters included choice of antibody construct, antibody density, antibody conjugation procedure, and choice of liposome construct. Anti-HER2 immunoliposomes bound efficiently to and internalized in HER2-overexpressing cells in vitro as determined by fluorescence microscopy, electron microscopy, and quantitative analysis of fluorescent probe delivery. Delivery via ILs in HER2-overexpressing cells yielded drug uptake that was up to 700-fold greater than with non-targeted sterically stabilized liposomes. In vivo, anti-HER2 ILs showed extremely long circulation as stable constructs in normal adult rats after a single i.v. dose, with pharmacokinetics that were indistinguishable from sterically stabilized liposomes. Repeat administrations revealed no increase in clearance, further confirming that ILs retain the long circulation and non-immunogenicity of sterically stabilized liposomes. In five different HER2-overexpressing xenograft models, anti-HER2 ILs loaded with doxorubicin (dox) showed potent anticancer activity, including tumor inhibition, regressions, and cures (pathologic complete responses). ILs were significantly superior vs. all other treatment conditions tested: free dox, liposomal dox, free MAb (trastuzumab), and combinations of dox+MAb or liposomal dox+MAb. For example, ILs produced significantly superior antitumor effects vs. non-targeted liposomes (P values from <0.0001 to 0.04 in eight separate experiments). In a non-HER2-overexpressing xenograft model (MCF7), ILs and non-targeted liposomal dox produced equivalent antitumor effects. Detailed studies of tumor localization indicated a novel mechanism of drug delivery for anti-HER2 ILs. Immunotargeting did not increase tumor tissue levels of ILs vs. liposomes, as both achieved very high tumor localization (7.0–8.5% of injected dose/g tissue) in xenograft tumors. However, histologic studies using colloidal-gold labeled ILs demonstrated efficient intracellular delivery in tumor cells, while non-targeted liposomes accumulated within stroma, either extracellularly or within macrophages. In the MCF7 xenograft model lacking HER2-overexpression, no difference in tumor cell uptake was seen, with both ILs and non-targeted liposomes accumulating within stroma. Thus, anti-HER2 ILs, but not non-targeted liposomes, achieve intracellular drug delivery via receptor-mediated endocytosis, and this mechanism is associated with superior antitumor activity. Based on these results, anti-HER2 immunoliposomes have been developed toward clinical trials. Reengineering of construct design for clinical use has been achieved, including: new anti-HER2 scFv F5 generated by screening of a phage antibody library for internalizing anti-HER2 phage antibodies; modifications of the scFv expression construct to support large scale production and clinical use; and development of methods for large-scale conjugation of antibody fragments with liposomes. We developed a scalable two-step protocol for linkage of scFv to preformed and drug-loaded liposomes. Our final, optimized anti-HER2 ILs-dox construct consists of F5 conjugated to derivatized PEG-PE linker and incorporated into commercially available liposomal doxorubicin (Doxil®). Finally, further studies of the mechanism of action of anti-HER2 ILs-dox suggest that this strategy may provide optimal delivery of anthracycline-based chemotherapy to HER2-overexpressing cancer cells in the clinic, while circumventing the cardiotoxicity associated with trastuzumab+anthracycline. We conclude that anti-HER2 immunoliposomes represent a promising technology for tumor-targeted drug delivery, and that this strategy may also be applicable to other receptor targets and/or using other delivered agents.

Introduction

We have developed anti-HER2 immunoliposomes (ILs) to combine the tumor-targeting properties of certain anti-HER2 monoclonal antibody (MAb) fragments with the pharmacokinetic and drug delivery advantages of long circulating liposomes. This approach has a number of potential advantages as compared with other immunoconjugate strategies. For example, in contrast to immunotoxins, liposomal agents can in principle be constructed so as to provide long circulation with minimal immunogenicity. Also, immunoliposomes can provide highly concentrated delivery of potent anticancer agents, and these encapsulated agents can possess their own inherent selectivity against cancer cells (e.g., accepted chemotherapeutic drugs), thus minimizing the potential for cytoxicity against normal tissues with low antigen expression. Finally, diffusion of drug released by immunoliposomes can effect bystander killing of nearby cancer cells, thus obviating the need for 100% targeting efficiency.

As a target antigen, HER2 is a readily accessible cell surface receptor, and, when overexpressed, provides a basis for selective immunotargeting of tumor cells. The HER2 (c-erbB-2, neu) protooncogene encodes a 1255 amino acid, 185 kDa receptor tyrosine kinase (RTK) that is a member of the class I RTK family, along with the epidermal growth factor receptor (EGFR), HER3 (ErbB3), and HER4 (ErbB4) (for a review, see Refs. [1], [2]). As an oncogene product, HER2 plays an important role in the development and progression of many breast and other cancers. HER2 is overexpressed in 20–30% of breast and ovarian cancers, most commonly via gene amplification, and overexpression is associated with poor prognosis in these patients [3], [4]. In addition to breast and ovarian cancer, HER2-overexpression also occurs frequently in a number of other carcinomas (for a review, see Ref. [5]). In normal adult tissues, HER2 occurs only at low levels in certain epithelial cell types [6]. When present in cancer, HER2-amplification and overexpression typically display a relatively homogeneous distribution within primary breast tumors, and also appear to be retained or increased at metastatic sites, suggesting a continuous requirement for HER2-overexpression throughout the malignant process [7]. This is in contrast to most other tumor-associated antigens, which are often heterogeneously expressed and/or readily down-modulated in tumor cells. Other studies have also implicated ErbB2 as a direct contributor to metastasis [8], [9], [10] and angiogenesis [11], further raising the possibility that ErbB2 expression may be upregulated or selected for during metastatic progression. For example, one study suggests that ErbB2 expression is highly prevalent (67%) in bone marrow micrometastasis of breast cancer patients [12].

Monoclonal antibodies directed against HER2 offer one strategy for targeted anticancer therapy. One such antibody, muMAb 4D5, specifically binds HER2 in its extracellular domain (ECD), and inhibits the growth of HER2-overexpressing breast cancer cells in vitro and in animal models [13], [14], [15]. A humanized version of this antibody, rhuMAb HER2 (trastuzumab, Herceptin®), was developed to retain these properties while reducing the potential for immunogenicity [16]. In clinical studies in advanced breast cancer, trastuzumab induced antitumor responses as a single agent [17], [18], and was particularly effective when combined with anthracycline- or taxane-based chemotherapy [19]. Based on these results, trastuzumab was approved by the FDA in 1998 for the treatment of advanced breast cancer, and thereby became the first MAb to achieve this milestone for solid tumor treatment. Next-generation strategies can be envisioned that combine the now validated approach of anti-HER2 MAbs with the drug delivery capability of liposomes.

Much like MAb technology, liposome technology required extensive optimization and clinical testing prior to attaining clinical validation. The first generation of liposomal pharmaceuticals, so-called ‘conventional’ liposomes, have been used to deliver a number of anti-cancer agents, sometimes resulting in reduced toxicity to normal tissues. However, a major limitation of conventional liposomes is their susceptibility to rapid clearance by the reticuloendothelial system (RES) [20]. The development of modified liposomes capable of prolonged circulation represents a significant advance in drug delivery. For example, so-called ‘stealth’ or ‘sterically stabilized’ liposomes (Ls) are small unilamellar particles of neutral charge, and contain an outer coat of inert polymers such as PEG (polyethylene glycol) [21], [22]. Sterically stabilized liposomes undergo much slower clearance by the RES, resulting in significant prolongation of circulation and markedly altered biodistribution. Importantly, sterically stabilized liposomes have been shown to preferentially accumulate in tumor tissue, and can reach significantly higher levels in tumor than in normal tissues, as shown in both preclinical and clinical studies [23], [24]. This property, an example of the ‘enhanced permeability and retention (EPR) effect’ [25], is believed to occur because of structural abnormalities in tumor-associated vessels that arise during tumor angiogenesis, which greatly facilitate liposome extravasation. These advances in liposome technology have generated renewed interest in liposomes as drug carriers [26]. Two liposomal agents (liposomal doxorubicin, Doxil®; liposomal daunorubicin, Daunosome®) have been approved by the US FDA for the treatment of AIDS-associated Kaposi’s sarcoma. In addition, sterically stabilized liposomal doxorubicin recently received approval for the treatment of refractory ovarian cancer.

Although capable of enhanced accumulation in tumor tissue, existing liposomal chemotherapeutics do not interact directly with tumor cells in vitro or in vivo, and instead release drug for eventual diffusion into tumor cells. Indeed, the same modifications that enable long circulation (e.g., steric stabilization) prevent liposome–cancer cell interactions. Immunoliposomes (ILs) represent a logical strategy to achieve direct drug delivery to tumor cells, by linking liposomes to monoclonal antibody fragments against tumor-associated antigens (for a review, see Ref. [27]). Thus, we have developed anti-HER2 ILs for targeted, intracellular drug delivery.

Section snippets

Preparation of anti-HER2 immunoliposomes (ILs)

Anti-HER2 immunoliposomes (ILs) were constructed by conjugation of anti-HER2 MAb fragments to sterically stabilized liposomes (Ls), to create a tumor-targeted drug delivery vehicle for the treatment of HER2-overexpressing breast cancers. Immunoliposome design involves multiple components [27], and ILs were constructed accordingly to optimize intracellular drug delivery.

Anti-HER2 ILs were prepared from small unilamellar liposomes containing 0–12 mol% PEG-phosphatidylethanolamine (PEG-PE; 0–12

Conclusions

We have developed anti-HER2 immunoliposomes (ILs) to combine the tumor-targeting properties of certain anti-HER2 monoclonal antibodies (MAbs) with the pharmacokinetic and drug delivery properties of long circulating liposomes (Ls). We have shown that anti-HER2 ILs efficiently bind to and internalize in HER2-overexpressing cells in vitro, resulting in intracellular drug delivery. In vivo, dox-loaded ILs (ILs-dox) displayed long circulation that was identical to that of sterically stabilized

Acknowledgments

This work was supported by grants from the National Cancer Institute Specialized Programs of Research Excellence (SPORE) in Breast Cancer (P50-CA 58207-01), the Department of Defense Breast Cancer Research Program Career Development Award (DAMD17-94-J-4195), the American Society of Clinical Oncology Young Investigator Award (J.W.P.) sponsored by the Don Shula Foundation, and the Janet Landsfears Fund of the Mt. Zion Health Fund.

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