Yingjuan Lu

Folate-mediated delivery of macromolecular anticancer therapeutic agents

The receptor for folic acid constitutes a useful target for tumor-specific drug delivery, primarily because: (1) it is upregulated in many human cancers, including malignancies of the ovary, brain, kidney, breast, myeloid cells and lung, (2) access to the folate receptor in those normal tissues that express it can be severely limited due to its location on the apical (externally-facing) membrane of polarized epithelia, and (3) folate receptor density appears to increase as the stage/grade of the cancer worsens. Thus, cancers that are most difficult to treat by classical methods may be most easily targeted with folate-linked therapeutics. To exploit these peculiarities of folate receptor expression, folic acid has been linked to both low molecular weight drugs and macromolecular complexes as a means of targeting the attached molecules to malignant cells. Conjugation of folic acid to macromolecules has been shown to enhance their delivery to folate receptor-expressing cancer cells in vitro in almost all situations tested. Folate-mediated macromolecular targeting in vivo has, however, yielded only mixed results, largely because of problems with macromolecule penetration of solid tumors. Nevertheless, prominent examples do exist where folate targeting has significantly improved the outcome of a macromolecule-based therapy, leading to complete cures of established tumors in many cases. This review presents a brief mechanistic background of folate-targeted macromolecular therapeutics and then summarizes the successes and failures observed with each major application of the technology.

Folate targeting of haptens to cancer cell surfaces mediates immunotherapy of syngeneic murine tumors.

Abstract.A variety of human cancers overexpress a cell surface receptor with high affinity for the vitamin, folic acid (Kd ~10–10 M). Covalent attachment of therapeutic agents to folic acid has been shown to allow efficient targeting of the folate–drug conjugates to folate receptor-expressing cancer cells, with little or no uptake by normal tissues except the kidneys. We report here the use of folates ability to deliver attached molecules specifically to cancer cells to convert poorly immunogenic tumors into highly immunogenic tissue targets. By linking folic acid to a model hapten, we have been able to decorate folate receptor-expressing cancer cell surfaces with >106 haptens/cell in vivo. Following marking of such cells with haptens, the cells are observed to become opsonized with autologous anti-hapten antibodies, which is presumed to mediate cell removal via antibody-dependent cellular cytotoxicity (ADCC). Supplemental administration of low levels of ADCC-activating cytokines [e.g. interleukin-2 (IL-2) and interferon-alpha (IFN-α)] has been shown to synergize with the folate-targeted immunotherapy. Thus, using M109 syngeneic lung cancer cells injected intraperitoneally into Balb/c mice that were previously immunized against fluorescein, a significant extension of life span is observed following treatment with folate–fluorescein conjugates, and complete cures are observed upon supplementation with moderate levels of IL-2 and IFN-α. Because control tumor-bearing mice treated with the same cytokines but with non-targeted fluorescein show no extension of life span, we conclude that tumor-specific opsonization is an essential step in this immunotherapy. Finally, because the anti-fluorescein antibodies are unable to access the folate receptors on the apical membranes of the kidney proximal tubules, no kidney or other normal tissue cytotoxicity is observed. These data suggest that retargeting of haptens to folate receptor-expressing cancers might constitute a method for mobilizing the immune system specifically against poorly immunogenic tumors.

Immunotherapy of folate receptor-expressing tumors: Review of recent advances and future prospects

The cell surface receptor for the vitamin folic acid (termed the folate receptor), is often elevated in cancers of the ovary, kidney, lung, mammary gland, brain, endometrium, and myeloid cells of hematopoietic origin. Because the folate receptor (FR) is either absent from normal tissues or localized to the apical surfaces of polarized epithelia, where it is inaccessible to circulating drugs, folate-linked drugs do not normally accumulate in healthy tissues. However, since the same receptor is fully accessible on cancer cells, it has frequently been exploited as a target for receptor-directed cancer therapies, including chemotherapies and immunotherapies. In fact, most strategies for the immunotherapy of cancer have at some time been adapted to treat FR-expressing tumors. In this article, recent progress in the retargeting of the immune system to folate receptor-expressing cancers is summarized and future strategies for redirecting natural killer cells, antibodies and cytotoxic T lymphocytes to this large class of malignancies are proposed.

Issues related to targeted delivery of proteins and peptides

While modern genomic and proteomic technology enables rapid screening of novel proteins and peptides as potential drug candidates, design of delivery systems for these biologics remains challenging especially to achieve site-specific pharmacological actions. This article discusses the issues associated with targeted delivery of protein and peptide drugs at physiochemical, physiological, and intracellular levels with a special focus on cancer therapy.

Folate receptor-targeted immunotherapy: Induction of humoral and cellular immunity against hapten-decorated cancer cells

We previously exploited the frequent overexpression of folate receptors on cancer cells to decorate malignant cell surfaces selectively with folate‐hapten conjugates. In antihapten‐immunized hosts, this targeted localization of foreign haptens to tumor cells led to rapid accumulation of autologous antihapten IgG, which in turn yielded potent antitumor activity upon stimulation with cytokines (IL‐2, IFN‐α). In an effort to understand the effector mechanisms responsible for tumor regression, we have now investigated the involvement of both humoral and cellular immune components in the tumor destruction process. We report that the dependence of therapeutic efficacy on folate‐hapten concentration is bimodal, suggesting that the conjugate must bridge between a cell surface FR and an antihapten IgG in order to mediate killing. Studies with cancer cells in vitro further demonstrate that folate‐fluorescein‐marked tumor cells are killed primarily by antibody‐dependent cellular cytotoxicity and phagocytosis, with no contribution from complement‐dependent mechanisms. Investigations of specific immune cell involvement also reveal that asialo‐GM1+‐natural killer cells, macrophages, CD4+ T cells and CD8+ T cells contribute significantly to recognition/removal of the cancer mass, and that elimination of these cell types markedly compromises the therapy. Because the initial antibody‐dependent stage of tumor cell killing is shown to lead to a long‐term antibody‐independent cellular immunity that involves both CD4+ and CD8+ T cells, we propose that Fc receptor‐expressing immune cells not only initiate destruction of the IgG‐marked tumor cells, but also participate in presentation of endogenous tumor antigens in a manner that leads to long‐term cellular immunity.

Treatment of experimental adjuvant arthritis with a novel folate receptor-targeted folic acid- aminopterin conjugate

IntroductionFolate receptor (FR)-expressing macrophages have been shown to accumulate at sites of inflammation, where they promote development of inflammatory symptoms. To target such a macrophage population, we designed and evaluated the biologic activity of EC0746, a novel folic acid conjugate of the highly potent antifolate, aminopterin.MethodsUsing a FR-positive subclone of murine macrophage-derived RAW264.7 cells and rat thioglycollate-elicited macrophages, we studied the effect of EC0746 on dihydrofolate reductase activity, cell proliferation, and cellular response towards bacterial lipopolysaccharide as well as IFNγ activation. The EC0746 anti-inflammatory activity, pharmacokinetics, and toxicity were also evaluated in normal rats or in rats with adjuvant-induced arthritis; that is, a FR-positive macrophage model that closely resembles rheumatoid arthritis in humans.ResultsEC0746 suppresses the proliferation of RAW264.7 cells and prevents the ability of nonproliferating rat macrophages to respond to inflammatory stimuli. In the macrophage-rich rat arthritis model, brief treatment with subcutaneously administered EC0746 is shown to mediate an FR-specific anti-inflammatory response that is more potent than either orally administered methotrexate or subcutaneously delivered etanercept. More importantly, EC0746 therapy is also shown to be ~40-fold less toxic than unmodified aminopterin, with fewer bone marrow and gastrointestinal problems.ConclusionsEC0746 is the first high FR-binding dihydrofolate reductase inhibitor that demonstrates FR-specific anti-inflammatory activities both in vitro and in vivo. Our data reveal that a relatively toxic anti-inflammatory drug, such as aminopterin, can be targeted with folic acid to inflammatory macrophages and thereby relieve inflammatory symptoms with greatly reduced toxicity.

Preclinical pharmacokinetics, tissue distribution, and antitumor activity of a folate-hapten conjugate–targeted immunotherapy in hapten-immunized mice

Folic acid (pteroylglutamic acid) represents a useful ligand for targeted cancer therapies because it binds to a common epithelial tumor antigen known as the folate receptor. We previously devised an immunotherapy strategy that uses a bispecific ligand, a folate-hapten (FITC) conjugate, to redirect endogenously induced anti-FITC antibodies to folate receptor–positive tumor cells following parenteral administration. Here, we present results from preclinical pharmacokinetic and tissue biodistribution studies using a radioactive folate-FITC conjugate and results from dose optimization studies done in tumor-bearing animals. Folate-FITC was found to be rapidly eliminated in non-immunized mice; however, in immunized hosts, folate-FITC was shown to form immune complexes with FITC-specific antibodies, the consequence of which was a ∼173-fold increase in drug exposure (i.e., area under the curve). Using a newly developed ELISA assay, the extent of circulating anti-FITC antibodies occupied by parenterally given folate-FITC was determined to be proportional to the given dose. Furthermore, high doses of folate-FITC were found to promote the cosaturation of tumor cell surface folate receptors and circulating FITC-specific antibodies, blocking the immune recognition of tumor cells and thereby reducing antitumor activity. Nonetheless, by extending the duration of treatment and administering subsaturating doses of folate-FITC, enhanced antitumor response was observed in mice bearing established folate receptor–positive M109 tumors. Overall, results from the present study may help to guide clinicians through on-going clinical investigations of folate-targeted immunotherapy. [Mol Cancer Ther 2006;5(12):3258–67]

A phase I study of folate immune therapy (EC90 vaccine administered with GPI-0100 adjuvant followed by EC17) in patients with renal cell carcinoma.

This is the first phase I, open-label study to assess the safety, pharmacokinetics, and antitumor activity of a novel immunotherapeutic regimen known as Folate Immune (EC90 vaccine administered with GPI-0100 adjuvant followed by EC17, a folate-targeted hapten immunotherapy that targets folate receptor expressing cancer cells), which is designed to convert poorly immunogenic tumors to highly immunogenic tumors in patients with metastatic renal cell carcinoma. Three to 6 patients were enrolled in each cohort. In the vaccination phase, patients were given once weekly vaccinations of 0.2 mg of EC90 plus 3.0 mg of GPI-0100 for 3–5 weeks. In the treatment phase, patients were treated with 0.031, 0.092, or 0.276 mg/kg of EC17, 5 d/wk, for weeks 3, 4, or 6. Forty-one patients were enrolled in the study of which 33 patients received ≥1 treatment of EC17. Two dose-limiting toxicities were observed including grade 4 anaphylaxis and grade 3 pancreatitis. During the vaccination phase, mild to moderate injection site reactions were the most frequently reported adverse events. During the treatment phase, transient hypersensitivity reactions were the most common adverse event. Partial response was noted in 4% (1/28) of patients, and stable disease was noted in 54% (15/28) of patients after cycle 1 and was maintained in the majority of patients entering the extension phase of the study. EC90 vaccine with GPI-0100 adjuvant followed by EC17 is safe and well tolerated. The recommended regimen for further studies is 4 weekly vaccinations with 0.2 mg of EC90 plus 3.0 mg GPI-0100 followed by treatment with 0.3 mg of EC17.

Synthesis and activity of folate conjugated didemnin B for potential treatment of inflammatory diseases.

A folate receptor targeted didemnin B conjugate was synthesized using a hydrophilic peptide spacer linked to folate via a releasable disulfide carbonate linker. Cell cytotoxicity and TNF-α inhibition in RAW264.7 macrophage-like cells exhibited IC(50)s of 13 and 5 nM, respectively. Folate didemnin B was found to be ∼50-100 fold more potent than didemnin B itself. More importantly, activity of the prodrug was blocked by excess folic acid, demonstrating receptor-mediated cellular uptake of the conjugate.

Strategy to Prevent Drug-Related Hypersensitivity in Folate-Targeted Hapten Immunotherapy of Cancer

Cancer vaccine/immunotherapy rarely involves systemic administration of an immunogenic compound to an actively immunized host. We have developed such a strategy that utilizes folate to deliver antigenic haptens [e.g., fluorescein (FITC) and dinitrophenyl] to folate receptor-positive tumors in a hapten-pre-vaccinated host. Here, we investigated the safety of this novel approach and developed strategies to prevent drug-related hypersensitivity. Using FITC as the model hapten, we identified a potential source of allergic species in folate–FITC preparations by LC-MS/MS. In mice and guinea pigs, we tested the significance of this impurity by passive cutaneous anaphylaxis and active systemic anaphylaxis assays. We studied the effect of immunogen (e.g., KLH–FITC) dose and derived a desensitization regimen that was further evaluated in a murine tumor model. Administration of folate–FITC with low multi-haptenated contaminants (e.g. bis-FITC) resulted in hypersensitivity in under-immunized animals. However, this drug-related hypersensitivity may be independently prevented by (1) increasing the immunogen dose and/or (2) desensitizing animals with folate–FITC during vaccination. In addition, such manipulation in vivo did not appear to negatively alter the effectiveness of immunotherapy. This study provided confidence on the safety of folate–hapten-targeted cancer immunotherapy in an actively immunized host.