The transferrin receptor part II: Targeted delivery of therapeutic agents into cancer cells

Clinical Immunology (2006) 121, 159 — 176

available at www.sciencedirect.com

www.elsevier.com/locate/yclim

REVIEW

The transferrin receptor part II: Targeted delivery of therapeutic agents into cancer cells Tracy R. Daniels a, Tracie Delgado a, Gustavo Helguera a, Manuel L. Penichet a,b,c,* a

Division of Surgical Oncology, Department of Surgery, David Geffen School of Medicine, UCLA, Los Angeles, CA 90095, USA Department of Microbiology, Immunology, and Molecular Genetics, David Geffen School of Medicine, UCLA, Los Angeles, CA 90095, USA c Jonsson Comprehensive Cancer Center, David Geffen School of Medicine, UCLA, Los Angeles, CA 90095, USA b

Received 7 June 2006; accepted with revision 16 June 2006 Available online 17 August 2006

KEYWORDS Transferrin receptor; Receptor-mediated endocytosis; Monoclonal antibodies; Recombinant antibodies; Targeted immunotherapy; Chemotherapy; Immunotoxin; Immunoconjugate

Abstract Traditional anti-cancer treatments consist of chemotherapeutic drugs that effectively eliminate rapidly dividing tumor cells. However, in many cases chemotherapy fails to eliminate the tumor and even when chemotherapy is successful, its systemic cytotoxicity often results in detrimental side effects. To overcome these problems, many laboratories have focused on the design of novel therapies that exhibit tumor specific toxicity. The transferrin receptor (TfR), a cell membrane-associated glycoprotein involved in iron homeostasis and cell growth, has been explored as a target to deliver therapeutics into cancer cells due to its increased expression on malignant cells, accessibility on the cell surface, and constitutive endocytosis. The TfR can be targeted by direct interaction with conjugates of its ligand transferrin (Tf) or by monoclonal antibodies specific for the TfR. In this review we summarize the strategies of targeting the TfR in order to deliver therapeutic agents into tumor cells by receptor-mediated endocytosis. D 2006 Elsevier Inc. All rights reserved.

* Corresponding author. Division of Surgical Oncology, Department of Surgery, UCLA 10833 Le Conte Avenue CHS 54-140 Mail code 178218, Los Angeles, CA 90095-1782, USA. Fax: +1 310 825 7575. E-mail address: [email protected] (M.L. Penichet). 1521-6616/$ — see front matter D 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.clim.2006.06.006

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Contents Introduction . . . . . . . . . . . . . . . . . . . . . Delivery of chemotherapeutic drugs . . . . . . . Doxorubicin (AdriamycinR ) . . . . . . . . . . Other drugs. . . . . . . . . . . . . . . . . . . Delivery of toxic proteins . . . . . . . . . . . . . Ricin. . . . . . . . . . . . . . . . . . . . . . . Other plant toxins . . . . . . . . . . . . . . . Fungal toxins . . . . . . . . . . . . . . . . . . Pseudomonas exotoxin . . . . . . . . . . . . Diphtheria exotoxin . . . . . . . . . . . . . . Ribonuclease . . . . . . . . . . . . . . . . . . Delivery of high molecular weight compounds . . Polymers/Polyplexes. . . . . . . . . . . . . . Tf conjugated liposomes . . . . . . . . . . . Single chain antibody conjugated liposomes Modified viral vectors . . . . . . . . . . . . . Nanoparticles. . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . .

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Introduction The TfR (also known as CD71), a type II transmembrane glycoprotein found as a homodimer (180 kDa) on the surface of cells, is a vital protein involved in iron homeostasis and the regulation of cell growth (recently reviewed in [1]). The TfR monomer contains a large extracellular C-terminal domain, a single-pass transmembrane domain, and a short intracellular Nterminal domain. The TfR is ubiquitously expressed on normal cells and expression is increased on cells with a high proliferation rate or on cells that require large amounts of iron [1]. Little or no TfR expression has been detected on pluripotent hematopoietic stem cells, while late erythroid and myeloid progenitor cells demonstrate TfR expression. Expression of the TfR is significantly upregulated in a variety of malignant cells and in many cases, increased expression correlates with tumor stage and is associated with poor prognosis [1]. Iron is involved in a variety of cellular processes and is a required co-factor for many enzymatic reactions including those involved in metabolism, respiration, and DNA synthesis [2,3]. Delivery and cellular uptake of iron occurs through the interaction and internalization of iron-loaded Tf mediated by the TfR [1] (Fig. 1). Tf is a monomeric glycoprotein (apo-Tf) that can transport one (monoferric Tf) or two (diferric Tf) iron atoms. Diferric Tf has the highest affinity for the TfR and is 10- to 100-fold greater than that of apo-Tf at physiological pH [3]. Upon binding the TfR, the Tf/TfR complex is internalized in clathrin-coated pits through receptor-mediated endocytosis. Due to the decrease in pH, iron is released from transferrin in the endosome. Tf remains bound to the receptor at this pH and the apo-Tf/TfR complex is recycled back to the cell surface where apo-Tf is then released. The TfR is constitutively recycled independently of Tf binding. A second transferrin receptor (TfR2) was identified and has a 25-fold lower affinity for Tf than TfR1 [1,4—6]. The human TfR2 a and h transcripts are produced by alternative splicing [6]. The TfR2 a and TfR1 only show similarity in their extracellular domains. In contrast to TfR1, TfR2a expression appears to be

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160 160 160 162 163 166 167 168 168 168 169 171 171 171 172 172 173 173 173 173

limited to hepatocytes and enterocytes of the small intestine and is not regulated by intracellular iron levels. High surface expression of TfR2a was detected in many solid and hematopoietic malignant human cell lines. The intracellular TfR2 h (lacks the transmembrane and cytoplasmic domains) is ubiquitously expressed at low levels and its function remains unclear. Traditional cancer therapy consists of chemotherapeutic drugs that can be successful in irradicating the tumor, but are often toxic to normal cells as well. Targeting the TfR is a promising strategy actively being explored as a drug alternative to offset these dangerous side effects. The high levels of expression of TfR in cancer cells, which may be up to 100-fold higher than the average expression of normal cells [7—9], its extracellular accessibility, its ability to internalize, and its central role in the cellular pathology of human cancer, make this receptor an attractive target for cancer therapy. In fact, the TfR can be successfully used to deliver cytotoxic agents into malignant cells including chemotherapeutic drugs, cytotoxic proteins, or high molecular weight compounds including liposomes, viruses, or nanoparticles (Fig. 2).

Delivery of chemotherapeutic drugs Doxorubicin (Adriamycin) R

Doxorubicin (AdriamycinR ) (ADR) is an anthracycline anticancer drug that blocks DNA synthesis and also blocks the activity of topoisomerase II, an enzyme that helps to relax the coil and extend the DNA molecule prior to DNA synthesis or RNA transcription. ADR is used to treat leukemia, breast cancer, and many other cancers. When used alone ADR often exhibits side effects including cardiotoxicity, myelosuppression, nephrotoxicity, and extravasation [10]. Systemic drug toxicity is often attributed to quick diffusion throughout the body resulting in a homogeneous tissue distribution [11]. The potential benefits of ADR may also be blocked by the development of drug resistant cancer cells. In an attempt to overcome these problems, delivery of ADR to the tumor by

Transferrin receptor

161

Figure 1 Cellular uptake of iron by the transferrin system via receptor mediated endocytosis. The TfR is constiuitively internalized via receptor-mediated endocytosis. Iron loaded Tf, if bound to the TfR homodimer, is internalized by endocytosis of clathrin-coated pits. The Tf/TfR complex is delivered into endosomes where the decrease in pH triggers the release of iron from the Tf/TfR complex. Iron is transported out of the endosome by the divalent metal ion transporter 1 (DMT1) and the Tf/TfR complex is recycled back to the cell surface. Tf dissociates from the receptor on the cell surface.

targeting the TfR has been extensively studied. The chemical conjugation of human Tf to ADR yields a conjugate that is toxic against a variety of human cell lines including Lovo (colorectal adenocarcinoma), HL-60 (leukemia cells), Hep2 (liver carcinoma), H-MESO-1 (mesothelioma), K562 (erythroleukemia), and HeLa (cervical adenocarcinoma) [12—16]. Due to the targeted delivery of ADR into tumor cells, the conjugate was 3to 10-fold more cytotoxic than ADR alone depending on the cell line. The Tf-ADR conjugate prolonged the lifespan of human HMESO-1 tumor bearing mice when compared to ADR alone or ADR plus free Tf (not conjugated) [12]. Tf-ADR was also shown to have altered trafficking properties when compared to Tf alone [17]. The binding properties and affinity of the Tf-ADR conjugate is half that of Tf alone while the rate of iron uptake by the cell is reduced. There is also an increase in the endocytosis and recycling time of the receptor. It has been suggested that internalization of the conjugate can occur by an undefined TfR-independent mechanism since a 100-fold excess of Tf could not completely block the cytotoxic effects of ADR in L929 mouse fibroblast cells [18] (Table 1). Tf-ADR conjugates were shown to be cytotoxic to other human cancer cell lines including MDA-MB-468 (breast cancer), U937 (leukemia), and LZFL 529 (large cell carcinoma) but only at levels comparable to free ADR [19]. Interestingly, the acid-sensitive Tf-ADR conjugates were significantly less toxic than free ADR in human umbilical vein endothelial cells (HUVEC). Although the conjugate did not increase the cytotoxic effects compared to ADR alone, the therapeutic window was increased to due lack of cytotoxicity in normal cells. Only acid-sensitive maleimide derivatives of the Tf-ADR conjugates were cytotoxic to these cancer cells, suggesting that disruption of the linkage between Tf and ADR is required for the observed ADR toxicity. Acid-sensitive ADR immunoconjugates consisting of the murine IgG1 anti-human TfR antibody (5E9) have also shown efficacy against human Daudi B lymphoma and Raji

Burkett’s lymphoma cell lines and xenograft models [20], while other acid-insensitive 5E9 immunoconjugates did not. This suggests that a pH-dependent step is required for cytotoxicity of the ADR conjugates in some cell lines. Conjugation of ADR with Tf could also serve as an alternative therapy by overcoming the resistance to ADR that develops in some malignancies. The Tf-ADR conjugate overcame resistance to ADR in human KB (oral carcinoma) cells [21]. Modified Tf-ADR conjugates reversed the resistance of MCF-7 (human breast cancer) ADR resistant cells [22]. Tf-ADR conjugates were saturated with either iron or the antineoplastic drug gallium nitrate (GN). GN shares certain chemical properties with iron and thus binds tightly to Tf [23]. GN is a competitive inhibitor of iron-bound Tf that disrupts iron homeostasis. By binding to Tf, GN blocks iron internalization and once inside the cell GN blocks DNA synthesis by directly binding and inhibiting the ribonucleotide reductase. This leads to cell death via the intrinsic apoptotic pathway. In ADR sensitive cells, ADR-GN-Tf conjugates exhibited the same effects as ADR alone. However in ADR-resistant cells ADR-GN-Tf overcame the resistance to ADR and decreased the IC50 (concentration needed to inhibit 50% of cell growth) by 100-fold. The ADR-Fe-Tf conjugate demonstrated a more modest increase in sensitivity to ADR, indicating a 10-fold increase in the inhibitory effect. Reversal of drug resistance was accompanied by a decrease in the multiresistance protein transcription level. The mechanism of action of ADR alone is by its intercalation into DNA and blocking the enzymatic activity of topoisomerse II [24]. In ADR-resistant cells, ADR was sequestered in cytoplasmic vesicles [22]. Interestingly, in GA-Tf-ADR treated cells ADR was localized to the nucleus where it presumably exerts its cytotoxic effects via its interaction with DNA. The GA-Tf-ADR conjugate (1) increased drug uptake by using Tf as a targeting molecule; (2) inhibited drug efflux by suppression of multiresistance protein gene

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Figure 2 Strategies used to target the TfR and deliver therapeutic agents to malignant cells. The TfR can be targeted by direct interaction with conjugates of its ligand Tf as well as by monoclonal antibodies or single chain antibody fragments targeting the extracellular domain of the TfR. Targeting the TfR has been utilized to deliver chemotherapeutic drugs, protein toxins, radionuclides, liposomes, modified viral particles, and nanoparticles.

expression; and (3) increased the amount of ADR in the nucleus. However, Tf-ADR conjugates may also target the plasma membrane [25,26]. The Tf-ADR conjugate exerts its cytotoxic effects by disrupting the transplasma membrane electron transport redox system, which is involved in cell growth, by blocking the activity of the NADPH dehydrogenase and oxidase located in the plasma membrane [14]. Therefore, TfR-ADR conjugates may also exert their cytotoxic effects via a DNA-intercalation independent mechanism.

Other drugs Other drugs have been conjugated to Tf in order to block the non-specific side affects observed by the drug alone. Cisplatin (Platinol-AQR ) is an alkylating agent that blocks DNA synthesis that is used to treat metastatic testicular and ovarian caner as well as advanced bladder carcinoma. Cisplatin chemically conjugated to the iron-binding site of Tf inhibits the growth of human adenocarcinoma and epidermoid cell lines [27,28]. These studies also showed in vivo efficacy of this conjugate by its ability to block the growth of mammary carcinoma in rats and melanoma growth in mice. Furthermore, Tf-cisplatin in combinations with ADR act synergistically to increase the cytotoxicity of either agent alone [29]. Phase I clinical trials demonstrated safety in patients and a 36% (4 of 11) response rate of patients with advanced breast cancer [30]. One complete response was observed. A second clinical trial demonstrated that the sequential treatment of iron chelators and Tf-cisplatin resulted in 87% (7 of 8) partial response rate in patients with advanced breast cancer [30].

Chlorambucil (LeukeranR ) is another alkylating agent that interferes with DNA replication and RNA transcription. It is used to treat chronic lymphoid leukemia, Non-hodgkin’s lymphomas, and advanced ovarian or breast tumors. Chlorambucil was chemically conjugated to Tf through a maleimide derivative [31]. Like the Tf-ADR conjugates, only acid sensitive Tf-chlorambucil conjugates showed increased cytotoxicity in MCF7 human breast cancer and MOLT4 human leukemia cell lines compared to the drug alone [31]. Preliminary toxicity studies demonstrated safety of the conjugate in mice. Two of the 3 chlorambucil treated mice died while all three of the conjugate treated mice survived. No significant weight loss was observed in the conjugate treated animals, indicating the absence of non-specific toxicities [31]. Mytomycin C (MutamycinR ) is another DNA alkylating agent that leads to extensive DNA crosslinking. Mytomycin C inhibits the synthesis and function of DNA and has been chemically conjugated to Tf using glutaraldehyde [31,32]. The binding capacity of the conjugate was similar to that of Tf and internalization was not changed by the conjugation. The rates of internalization of the conjugate in the human hepatoma HepG2 cell line and normal hepatocytes were also similar. However, larger amounts of the conjugate were taken up by HepG2 cells presumably due to their higher level of cell surface TfR expression. The conjugate significantly inhibited the growth of HL-60 cells, but only slightly inhibited the growth of HepG2 cells when compared to Mytomycin C by itself. No toxicity was observed in normal hepatocytes treated with the conjugate. Other drugs conjugated to Tf have been developed and studied for their potential anti-tumor effects. Gemcitabine

Transferrin receptor Table 1

163

Delivery of chemotherapeutic drugs by human Tf or anti-TfR antibodies

Conjugate Human Tf Tf-ADR

Tf-GA-ADR Tf-Fe-ADR Tf-Cisplatin

Tf-chlorambucil

Tf-MMC Tf-Gemcitabine Tf-daunorubicin Anti-human TfR 5E9-ADR

In vitro cytotoxicity Human leukemia (HL-60) Erythroleukemia (K562)

Human colorectal carcinoma (Lovo) Human breast adenocarcinoma (MDA-MB-468) Human leukemia (U937) Human large cell carcinoma (LXFL 529) Human mesothelioma (H-MESO-1), Human liver carcinoma (Hep2) Human cervical adenocarcinoma (HeLa) Human cervical carcinoma ADR-resistant (KB) Mouse fibroblast (L929) Human breast (MCF-7) Human adenocarcinoma and epidermoid cells

Human breast (MCF-7) Human leukemia (MOLT-4) Human leukemia (HL-60) Human hepatoma (HepG2) Human bladder carcinoma (UCRU-BL13 and UCRU-BL28) Human small cell lung carcinoma (NCI-H69)

Human Daudi B lymphoma and Raji Burkitt’s lymphoma

(GemzarR ) is an anti-metabolite pyrimidine analog that blocks DNA synthesis. Gemcitabine has been chemically linked to Tf via a carbohydrate moiety and the resulting conjugates demonstrated a 6-fold increase in cytotoxicity to UCRU-BL13 and UCRU-BL28 human bladder cancer cells compared to drug alone [33]. This conjugate also showed therapeutic activity by partial remission of UCRU-BL-28 xenografts in SCID mice. Daunorubicin (CerubidineR ) is an anthracycline topoisomerse inhibitor that also blocks DNA synthesis and RNA transcription. Chemical conjugation of Tf to daunorubicin using glutaraldehyde resulted in a conjugate that inhibited growth of the NCI-H69 small cell lung carcinoma cell line 10-fold when compared to daunorubicin alone [34].

Comments

Conjugate binding affinity is about half of Tf Decreases the level of iron uptake by the cells Increases the endocytosis rate and recycling time of Tf DNA-intercalation independent mechanism of ADR ADR targets the plasma membrane Acid sensitive conjugates demonstrate toxic effects

References [12,13,15] [13,15,16]

[25] [26] [12] [19]

Prolongs the lifespan of nude mice bearing xenografts Blocks electron transport at the plasma membrane Overcomes ADR resistance

[12]

[21]

Overcomes ADR resistance Overcomes ADR resistance

[18] [22]

Blocks mammary carcinoma growth in rats Blocks melanoma growth in mice Phase I Clinical Trial (36% rate in advanced breast cancer patients) Clinical Trial (87% partial response rate in advanced breast cancer patients treated with iron chelators followed by Tf-cisplatin) Acid-sensitive conjugates demonstrated toxic effects Toxicity studies demonstrated safety in mice No toxicity observed in normal hepatocytes

[27,28]

[14]

[30] [30]

[31]

[32]

6-fold more cytotoxic than free drug Partial remission of xenografts in SCID mice 10-fold more cytotoxic than free drug

[33]

Acid-sensitive conjugates demonstrated toxic effects; Anti-tumor effects also observed in nude mice bearing Daudi B lymphoma xenografts

[20]

[34]

Delivery of toxic proteins In addition to chemotherapeutic drugs, the TfR has been used for the targeted delivery of toxic proteins into malignant cells. An immunotoxin describes a cell-specific ligand linked to a plant or bacterial toxin or modified toxin subunit [35,36]. The cell-specific ligand can either be an antibody, antibody fragment, cytokine, or other ligand that binds specifically to target cells and results in the internalization of the immunotoxin [35,36]. The TfR has been targeted by many immunotoxins to deliver plant, bacterial, or fungal toxins into tumor cells (summarized in Tables 2 and 3).

164 Table 2

T.R. Daniels et al. Delivery of toxic proteins by anti-TfR antibodies

Conjugate

Isotype

In vitro cytotoxicity

Comments

References

Ricin B3/25-RTA Anti-human TfR

IgG1

Human T-Leukemia (CCRF-CEM), cervical adenocarcinoma (HeLa), and melanoma (M21) 3D tumor model of human breast cancer (MCF7) spheroids Human Glioma cell lines and primary glioma cells Human mesothelioma (H-MESO-1)

Inhibited of HeLa and M21 xenograft growth.

[40]

Dose-dependent growth of inhibition.

[41]

OKT9-RTA Anti-human TfR 7D3-RTA Anti-human TfR

IgG1 IgG1

7D3-RTA plus ADR

454A12-RTA Anti-human TfR

H-MESO-1

IgG1

Human medulloblastoma, glioblastoma cell lines and primary cells isolated from patients, human breast cancer cell lines Human meduloblastoma, glioblastoma, and neuroblastoma and pediatric brain tumor specimens

Human ovarian cancer

OX26-RTA Anti-rat TfR

IgG2a

R17 217-RTA Anti-mouse TfR

Ig2a

Other plant toxins 42/6-saporin

IgA

B3/25-saporin

IgG1

3D tumor model of rat glioblastoma (9L) spheroids Murine T lymphoma cell line (BW5147), mouse myeloid and erythroid bone marrow progenitors

Primary glioma cells isolated from patients Human cell lines (K562 and HL-60), and primary cells from AML patients

[42] Increased survival in nude mice bearing H-MESO-1 xenografts. Cytotoxicity blocked by excess antibody. Mononesin potentiates cytotoxicity. Significantly increased survival of H-MESO-1 bearing nude mice. Toxicity studies showed safety in guinea pigs and rhesus monkeys.

1000-fold more cytotoxic than RTA expression level of TfR correlated with sensitivity to the immunotoxin. 30% reduction in mean tumor volume of U251 MG human glioma tumors established in nude mice. Cytotoxicity augmented by verapamil. Inhibits growth of NIH:OVCAR-3 xenografts in nude mice. Cytotoxicity blocked by excess Ab. Clinical trial (Toxin delivered to via IV injection) 4 of 8 patients showed more than a 50% reduction of tumor cell counts in lumbar CSF N 120 Ag local inflammatory response Dose-dependent growth of inhibition. Conjugate is cytotoxic to late erythroid progenitor cells in mouse bone marrow.

[39]

[44] [45]

[46]

[47]

[49]

[46] [45] [49] [53]

[41] [54]

[58] No toxicity observed with saporin alone or non-targeting immunotoxin.

[59]

Transferrin receptor

165

Table 2 (continued) Conjugate

Isotype

In vitro cytotoxicity

Comments

References

Other plant toxins 5E9-gelonin

IgG1

Various human leukemia and cervical

[62]

IgG1

Human lymphoblastic leukemia

Delayed growth and prolonged the survival of nude mice bearing Burkitt’s lymphoma xenografts. Cytotoxicity potentiated by the addition of chloroquine.

IgG1

Human T lymphoblastic leukemia (HSB-1)

[64]

Fungal toxins HB21-restrictocin or HB21 (Fv)-restrictocin

IgG1

[65—67]

HB21-a-sarcin

IgG1

Human T cell leukemia (HUT102), lung carcinoma (A549), breast cancer (MCF-7), and epidermoid carcinoma (A431) Human T cell leukemia (HUT102), lung carcinoma (A549), and breast carcinoma (MCF-7)

Pseudomonas exotoxin HB21-PE

IgG1

scFv(HB21)-PE40

IgG1

scFv(HB21)-LysPE40

IgG1

Various human cancer cell lines

Diphtheria exotoxin B3/25-DT catalytic domain

IgG1

[40]

454A12-CRM 107

IgG1

Human T-Leukemia (CCRF-CEM) and adenocarcinoma (HeLa) Various human cancer cell lines

Ribonuclease 42/6-bovine RNAse B3/25-bovine RNAse ScFv E6-human EDN

IgA IgG1 IgG1 chimera

Human erythroleukemia (K562) Human erythroleukemia (K562) Human erythroleukemia (K562) and epidermoid carcinoma (A431)

[79] [79] [81]

ScFv E6-human angiogenin

IgG1 chimera

E6 F(ab’)2-human angiogenin E6 CH2-human angiogenin

IgG1 chimera IgG1 chimera

Human breast adenocarcinoma (MDA-MB-231, colon adenocarcinoma (HT-29), and renal carcinoma (ACHN) Human erythroleukemia (K562) Human breast adenocarinoma (MDA-MB0231) and glioma (SF530)

5E9-pokeweed anti-viral protein 5E9-Luffa toxin

Human ovarian cancer (A1847) and breast carcinoma (MCF-7) cell lines and primary cells isolated form ovarian metastatic lesions Various human cancer cell lines including breast, ovarian, adult T-cell leukemia, colon and prostate)

[63]

Cytotoxicity blocked by the addition of excess HB21.

[68]

Cytotoxicity enhanced by verapamil.

[52,69]

Cytotoxicity blocked by the addition of excess HB21. KM12L4 cells growing in the liver or subcutaneously in nude mice (organ environment modulates TfR expression and determines the sensitivity to the fusion protein). Resulted in the regression of human epidermoid carcinoma (A431) subcutaneous xenografts in nude mice.

[9,70]

[71]

[46,47]

E6 does not compete with Tf. Fusion protein contains only 6—13% RNAse activity compared to EDN Fusion protein was 6-fold less in RNAse activity compared to angiogenin.

Angiogenin in the fusion protein remained fully active.

[82]

[83] [84]

166 Table 3

T.R. Daniels et al. Delivery of toxic proteins by human Tf

Conjugate

In vitro cytotoxicity

Comments

References

Tf-RTA

Leukemia CEM cells

[39]

Tf-RTA

3D tumor model of human breast cancer (MCF7) or rat glioblastoma (9L) spheroids Human erythroleukmia cell line (K562) Human hepatoma cell line (HepG2) Primary human glioma cells isolated from patients Mouse thymidine kinase-deficient L cells 3D tumor model of spheroids of MCF7 or rat glioblastoma 9L cells Various human cancer cell lines

10,000-fold increase in toxicity compared to RTA. Dose-dependent growth of inhibition.

Tf-saporin

Tf-DT Tf-DT

Tf-CRM 107

Primary cells isolated from patients with aggressive brain tumors

Tf-CRM 107 plus chloroquine

Tf-bovine RNAse

Human erythroleukemia (K562) cell line

Ricin The ricin toxin, derived from the seed of the Ricinus communis plant, is a ribosomal inactivating protein that is composed of two chains connected by a disulfide bond [37,38]. The A chain (RTA) contains the N-glycosidase enzyme that blocks ribosomal activity, while the B chain (RTB) is responsible for binding to the cell surface. The RTA chain by itself is not toxic due to the lack of cell binding ability, however, cell binding via targeting of the TfR restores the toxic affects of the A chain. Conjugation of human Tf with RTA increased the cytotoxic effects of RTA alone 10,000-fold [39]. This cytotoxicity was blocked by competition with free transferrin, antibodies to RTA, or antibodies to human Tf. The first report to examine immunotoxins targeting the TfR surfaced in the early 1980s [40]. The cytotoxic effects of the B3/25 murine anti-human TfR IgG1 antibody conjugated to RTA or the bacterial diphtheria toxin (to be discussed below) were examined. The CCRF-CEM T leukemic and HeLa cervical adenocarcinoma human cell lines were specifically killed by the B3/25-RTA immunotoxin. In vivo studies carried out in nude mice showed that one intravenous injection of B3/25 antibody alone inhibits growth of human melanoma M21 xenografts, while inhibition of cell growth was further

[41]

[56] [57] Cytotoxicity dependent on iron saturation of Tf. Cytotoxicity blocked by free Tf, anti-TfR, or ammonium chloride. Dose-dependent growth of inhibition.

Cytotoxicity blocked by the addition free transferrin or human TfR. Toxicity studies showed safety in guinea pigs and rhesus monkeys. Inhibits the growth of established U87 MG glioma xenografts in nude mice. Phase I and II clinical trials (malignant brain tumors that were refractory to conventional therapies). Chloroquine blocks toxicity without reducing anti-tumor effect in nude mice bearing U251MG human glioblastoma xenografts. RNase must be conjugated to Tf to show cytotoxic effect.

[58] [72] [41]

[46,47]

[50]

[75,76]

[78]

[79]

enhanced when mice were treated 3 times with B3/25-RTA. Multicellular tumor spheroids of either human breast cancer MCF7 cells sensitive to anti-TfR treatment or rat glioblastoma 9L cells (resistant) were created as a 3D tumor model that could be used to test the efficacy of Tf or anti-TfR conjugated to RTA [41]. Tf-RTA or murine monoclonal antibodies (anti-human TfR IgG1 OKT9 or anti-rat TfR IgG2a OX26) demonstrated anti-tumor activity in the 3D culture models. Response to treatment with high doses of any of the conjugates showed complete growth inhibition, although low doses showed heterogeneous responses [41]. Another immunotoxin, a conjugate of the murine monoclonal anti-human TfR IgG1 antibody 7D3 and RTA linked via a disulfide bond, demonstrated in vitro toxicity in H-MESO-1 cells (human malignant mesothelioma cells) and various human glioma cell lines including primary cells isolated from glioma patients [42,43]. The effect was blocked in H-MESO-1 cells by excess antibody, confirming that the cytotoxic effect is mediated through the TfR. In vivo analysis of 7D3-RTA treatment showed increased survival of nude mice bearing HMESO-1 xenografts, while irrelevant control immunotoxins showed no effect [43]. The in vitro and vivo effects of 7D3RTA can be enhanced by the addition of the carboxylic ionophore monensin, a known potentiator of immunotoxin

Transferrin receptor function [42,44]. Combination treatment of the 7D3-RTA conjugate and ADR demonstrated no change in H-MESO-1 cytotoxicity when compared to 7D3 alone [45]. However, in vivo studies of H-MESO-1 flank tumors established in nude mice showed that 7D3-RTA or drug alone increased the mean survival from 10 days in the control mice to 22—23 days. 7D3RTA in combination with ADR further increased the survival time to 31 days, which could be prolonged to 41 days when monensin was added as a third component. This study provides evidence that using conjugates in combination with traditional chemotherapeutic drugs could be an effective therapy for the treatment of certain malignancies. The therapeutic potential of a third ricin immunotoxin targeting the TfR through the use of the murine monoclonal IgG1 anti-human TfR 454A12 antibody has also been demonstrated in vitro. The 454A12-RTA conjugate is cytotoxic to human medulloblastoma, glioblastoma, neuroblastoma, and breast cancer cell lines as well as primary cells isolated from aggressive brain tumors but not to cells isolated from benign or non-aggressive tumors. [46,47]. The conjugate demonstrated no effect on U87 MG human glioma bearing nude mice [48], while it did reduce the mean U251 MG human glioma tumor volume by day 14 in a nude mouse model [49]. Tumor regrowth occurred within 10 days, but the tumor volume of 454A12-RTA treated mice remained lower than that of tumors in control animals. The 454A12-RTA immunotoxin is also cytotoxic to human ovarian cancer cells [50,51]. Verapamil, a calcium channel blocker, augments the cytotoxic effect of 454A12-RTA in human ovarian carcinoma cell lines [52]. 454A12-RTA blocked protein synthesis in the OVCAR-3 human ovarian carcinoma cell line, an effect that could be enhanced by the addition of recombinant human interferon a [50]. Intraperitoneal administration of 454A12RTA prolonged the mean survival time by 30% in nude mice bearing OVCAR-3 xenografts. Survival time was further increased to 89% when the immunotoxin was administered in combination with recombinant human interferon a. The efficacy of 454A12-RTA delivered to human patients has also been evaluated [53]. Intraventricular infusion [direct injection into the ventricles (cerebrospinal fluid filled cavities) of the brain] at low concentrations of the 454A12-RTA demonstrated no neural or systemic toxicity. Higher concentrations (120 Ag or more) resulted in an inflammatory response with the development of headache, vomiting, and altered mental status. However, tumoricidal concentrations of the immunotoxin were attained safely in the cerebrospinal fluid (CSF) of patients with leptomeningeal tumor spread. Four of 8 patients showed a greater than 50% reduction of tumor cell counts in lumber CSF within 5 to 7 days of treatment, but tumor clearance was never achieved. A rat anti-murine TfR IgG2a antibody (R17 217) conjugated to RTA demonstrated anti-proliferative effects in murine T-lymphoma cells BW5147, but not human CCRFCEM T-leukemic cells demonstrating species specificity [54]. Only a short exposure (15 min) to the R17 21-RTA conjugate was required for this cytotoxicity in the murine T-Lymphoma cell line. R17 217-RTA was also cytotoxic to late erythroid progenitor cells in the mouse, but less than 10% of pluripotent stem cells or early erythroid progenitors were inhibited under similar conditions. This suggests that R17 217-RTA may be used for therapeutic purposes without significant damage to the hematopoietic lineage [54].

167

Other plant toxins Saporin, a toxin derived from the plant Saponaria officinalis, is a ribosome-inactivating toxin that blocks protein synthesis [55]. The natural saporin toxin does not contain a cellbinding chain and is unable to efficiently bind the cell surface, making it an attractive alternative to modified forms of two chain toxins. Like Ricin, saporin inhibits protein synthesis through its N-glycosidase activity by inactivating the 28S ribosomal subunit. A Tf-saporin conjugate demonstrated cytotoxic effects on K562 (human erythroleumia) and HepG2 (human hepatoma) cells in vitro [56,57]. K562 cells treated with the combination of Tf and saporin (not conjugated) did not demonstrate growth inhibition. Tfsaporin was also cytotoxic to primary cells isolated from glioma patients, but was dependent upon iron saturation of Tf [58]. An immunotoxin consisting of the murine monoclonal anti-human TfR IgA 42/6 antibody and saporin was equally effective in primary glioma cells and was not dependent on iron saturation. Conjugation of the B3/25 antibody to saporin via a disulfide linkage blocked clonogenic growth of K562 and HL-60 cells after 48—72 h of treatment [59]. Saporin alone or a non-targeting immunotoxin showed no growth inhibition. We previously reported the development of two mouse/ human chimeric antibodies that were derived from either the variable region of the murine monoclonal anti-rat TfR IgG2a antibody OX26 or the variable region of the murine monoclonal anti-human TfR IgG1 antibody 128.1 [60]. Both contain the human IgG3 Fc region genetically fused to chicken avidin. These fusion proteins were designed as a universal delivery system to transport biotinylated agents into malignant cells. The anti-rTfR IgG3-Av delivered biotinylated FITC and h-galactosidase that remained biologically active inside the tumor cells. Surprisingly these antibody fusion proteins also demonstrated cytotoxic activity by themselves against certain tumor cell lines. In addition, this cytotoxic effect can be enhanced by the conjugation to biotinylated therapeutic agents. Preliminary studies from our group indicate that complexing of anti-hTfR IgG3-Av to biotinylated saporin significantly enhanced the intrinsic cytotoxic activity of the antibody fusion protein in malignant B and plasma cells [61]. Further studies are being conducted in our laboratory to determine the full therapeutic effect of this antibody fusion protein. Other immunotoxins consisting of single chain plant toxins have demonstrated growth inhibitory effects on human malignancies. Gelonin, derived from the seed of the Gelonium multiflorum plant, also lacks a cell-binding domain in its natural state and inactivates the 28S ribosomal subunit through its N-glycosidase activity. A conjugate of 5E9 and gelonin was highly toxic to human cancer cell lines, including Burkitt’s lymphoma, adult T cell acute lymphocytic leukemia, acute myelogenous leukemia, promyelocytic leukemia, and cervical carcinoma cell lines [62]. A murine leukemic cell line was not sensitive to the effects of the 5E9gelonin immunotoxin demonstrating species specificity. In vivo, this 5E9-gelonin was able to prolong the survival of nude mice bearing human Burkitt’s lymphoma xenografts [62]. The pokeweed anti-viral protein is another ribosomeinactivating protein derived from various parts of the Phytolacca Americana plant. It is also only composed of the enzymatic chain, and when chemically conjugated to

168 5E9 by a disulfide bridge, was cytotoxic to acute human lymphoblastic leukemia cells [63]. 5E9 has also been conjugated to a toxin isolated from the seeds of the Luffa aegyptiaca plant [64]. This toxin is a ribosomal inhibitory protein that is cytotoxic to the HSB-1 human T lymphoblastic leukemic cell line when conjugated to 5E9.

Fungal toxins Restrictocin, derived from the fungus Aspergillus restrictus, is another ribosome inactivating toxin. It is a single chain toxin that does not contain a cell-binding domain and is poorly immunogenic. Immunotoxins consisting of the murine monoclonal HB21 anti-human TFR (IgG1) antibody chemically conjugated to restrictocin inhibited protein synthesis in HUT102 (T-cell leukemia), A549 (lung carcinoma), MCF-7 (breast cancer), and A431 (epidermoid carcinoma) human cell lines [65]. This effect was only observed in cells treated with immunotoxins containing a cleavable linker between the antibody and restrictocin and was blocked by the addition of excess unconjugated antibody. Genetic fusions between the variable region of the HB21 antibody and restrictocin also inhibited protein synthesis in K562 (erythroleukemia), HUT102, A549, COLO205 (colon carcinoma), MCF-7, and A431 human cell lines [66]. These effects could be enhanced by the addition of a cleavable linker between the antibody fragment and restrictocin [67]. The HB21 antibody has also been conjugated to another fungal toxin, a-sarcin that is secreted by the Aspergillus giganteus fungus [68]. a-sarcin is a ribosomal inhibitory protein that interacts with 28S ribosomal subunit to block protein synthesis. This immunotoxin blocked protein synthesis in various human cancer cell lines including HUT102, U937 histocytic lymphoma, A549, and MCF-7. This effect could be blocked by the addition of excess HB21 antibody, indicating that targeting through the TfR is essential for the observed cytotoxic effects.

Pseudomonas exotoxin Bacterial toxins have also been delivered to tumor cells via the TfR. The pseudomonas exotoxin (PE) is derived from the bacterium Pseudomonas aeruginosa. PE is composed of three domains: an N-terminal catalytic domain, a translocation domain, and the C-terminal cell-binding domain [37]. The catalytic domain is an ADP-ribosyl transferase that blocks protein synthesis by ADP-ribosylating and inactivating elongation factor 2 that is required for protein synthesis. Cytotoxicity of the human cell lines A1847 (ovarian cancer) and MCF-7 was observed with the treatment of an immunotoxin consisting of HB21 and PE [69]. Ovarian cancer cell lines freshly established from metastatic lesions of four human ovarian lesions were also sensitive to HB21-PE. The level of growth inhibition varied among the cell lines and correlated with the levels of binding and internalization of the immunotoxin. Cytotoxicity of HB21-PE in ovarian cancer cell lines could be enhanced when used in combination with verapamil, a calcium channel blocker [53,69]. PE40 is a truncated form of the PE toxin that lacks the cell-binding domain. The truncated toxin has been used to create a single chain anti-TfR (scFv)-PE40 immunotoxin [9,70]. The anti-TfR (scFv)-PE40 immunotoxin consists of a

T.R. Daniels et al. genetic fusion between a single chain of the variable region of the HB21 antibody and PE40 [9,70]. The anti-TfR (scFv)PE40 fusion protein inhibited the growth of a variety of malignant human cell lines (including breast, ovarian, adult T-cell leukemia, colon, and prostate cancer) [70]. The cytotoxic affects of the anti-TfR (scFv)-PE40 could be blocked by pre-incubation of cells with excess HB21 antibody. The anti-TfR (scFv)-PE40 immunotoxin was generally more active compared to a similar immunotoxin consisting of anti-TfR (scFv) genetically fused to a truncated form of the diphtheria toxin. Depending on the cell line the anti-TfR (scFv)-PE40 immunotoxin was up to several hundred fold more active. The genetically conjugated anti-TfR (scFv)-PE40 was more potent than the chemical conjugation of the HB21 antibody with PE40. The effects of organ environment on TfR expression and sensitivity to the antiTfR (scFv)-PE40 conjugate have been examined in vivo [9]. The anti-TfR (scFv)-PE40 immunotoxin was also cytotoxic to the KM12L4 human colon carcinoma cell line. These cells were grown in the liver or subcutaneously in nude mice, after which a systemic intravenous administration of the anti-TfR (scFv)-PE40 immunotoxin eliminated the growth of liver metastases while a control antibody immunotoxin, targeting the Lewis-Y-related carbohydrate antigen did not. However, the anti-TfR (scFv)-PE40 conjugate only delayed the growth of the subcutaneous tumors, although tumor cells in the liver expressed higher levels of TfR than those grown subcutaneously. These data suggest that organ environment modulates TfR expression and as a result determines the sensitivity of the cells to the conjugate. Lys PE40 is a modified version of the PE40 toxin that has an extra lysine reside in the N-terminus that potentiates binding of the toxin to an antibody [71]. This lysine modified, truncated toxin has been used to create antibody immunotoxins that target the human TfR [71]. The anti-TfRLysPE40 immunotoxin was constructed by chemically conjugating the HB21 anti-human TFR (IgG1) antibody to LysPE40. The HB21-LysPE40 immunotoxin was cytotoxic to a variety of cells lines. Excess HB21 antibody blocked this cytotoxicity, providing evidence that targeting of the TfR is necessary for the effects observed. HB21-LysPE40, given four times over 8 days by intraperitoneal injection to nude mice, caused regression of established subcutaneous A431 epidermoid carcinoma tumors [71].

Diphtheria exotoxin Like PE, the bacterial diphtheria toxin (DT) that is secreted by Corynebacterium diphtheria, blocks protein synthesis by ADP-ribosylating elongation factor 2. DT is also composed of three domains: an N-terminal catalytic domain, a translocation domain, and the C-terminal cell-binding domain. Early studies on immunotoxins targeting the TfR performed in the early 1980s examined the effects of RTA (described above) and DT immunotoxins [40]. The B3/25 antibody conjugated to the DT enzymatic domain was cytotoxic to CCRF-CEM and HeLa human cell lines. Tf has also been covalently conjugated to the intact DT. The Tf-DT conjugate was highly cytotoxic to thymidine kinase-deficient mouse L cells in vitro [72]. This cytotoxic effect was blocked entirely by free Tf, anti-Tf, and anti-DT antibodies. Increasing the pH of intracellular compartments by the addition of ammonium chloride also

Transferrin receptor blocked the cytotoxic affect of Tf-DT. This suggests that the pH within the endosomal compartment is important for the release of the toxin from the organelle into the cytoplasm. CRM107 is a mutant form of the DT that contains two point mutations in the binding subunit (B chain) of the toxin resulting in the inability of CRM107 to bind the cell surface [73]. CRM107 alone shows a dramatic loss, at least 1000-fold, of toxicity compared to the wild type DT toxin. However, conjugation of CRM107 to a monoclonal antibody specific for human T-cells [73] or to Tf [74] restores the cytotoxic effect of the mutant. 454A12 conjugated to CRM107 and Tf-CRM107 (TransMID, Xenova Group) conjugates were cytotoxic to medulloblastoma, glioblastoma, neuroblastoma, leukemia, ovarian cancer and breast human cancer cell lines, as well as primary medulloblastoma cells isolated from patients [46,47]. CRM107 immunotoxins were found to kill at a faster rate compared to the corresponding RTA immunotoxins and CRM107 alone [46]. The maximum tolerable dose of Tf-CRM 107 was determined in vivo by intrathecal injection (injection into the CSF-filled space around the spinal cord) into guinea pigs and rhesus monkeys and found to be well within the therapeutic dose determined by in vitro analysis. It is important to note that the CSF was found to be an immunoprivileged site so anti-DT antibodies developed as a result of vaccination should not interfere with the activity of Tf-CRM107 in CSF. Tf levels in the CSF were not high enough to block the activity of Tf-CRM107. The efficacy of the Tf-CRM107 has also been evaluated in vivo in nude mice bearing established human U251 glioma xenografts [49]. Intratumoral injection of CRM107 alone caused significant U251 MG tumor growth inhibition, however, injection of Tf-CRM107 enhanced these growth inhibitory effects about 100-fold. Tf-CRM107 caused tumor regression in all of the treated mice (n = 5). The mean decrease in tumor volume was greater than 95% by day 14, and by day 30 there was still no evidence of tumor in 60% of the treated mice. No weight loss was observed by mice treated with Tf-CRM107, indicating that the immunotoxin was well tolerated. Primary cells isolated from patients with more aggressive tumors such as medulloblastoma and glioblastoma multiforme were susceptible to Tf-CRM107, whereas low-grade tumors were not sensitive [49]. Expression levels of TfR in cells isolated from patients was shown to correlate with sensitivity to these immunotoxins. A Phase I clinical trial of the Tf-CRM107 was initiated to determine the clinical relevance of this immunotoxin [75,76]. Tf-CRM107 was delivered by intratumoral infusion to patients with malignant brain tumors refractory to conventional therapies. Eighteen patients with various forms of brain tumors (10 glioblastoma multiforme, 5 anaplastic astrocytoma, 1 anaplastic oligodendroglioma, and 2 lung cancer metastasis) were treated with Tf-CRM107. A 50% decrease in tumor volume was observed in 9 of 15 (60%) patients, 2 of which demonstrated complete tumor regression. Sera from 43% patients showed a 2-fold increase in antidiphtheria antibody titers, which did not appear to predict response to Tf-CRM107. No systemic cytotoxicity was observed at any concentration. Local toxicity consistent with brain capillary endothelial damage (microvascular occlusion or petechial hemorrhage) was observed at higher doses of the conjugate (N 1 Ag/mL) in all three patients tested. The Tf-CRM107 conjugate did not cause any local toxicity in

169 patients at lower doses (b 1 Ag/mL) while anti-tumor activity was still evident. Systemic intraperitoneal injection of chloroquine, an antimalarial drug known to block the action of DT in vitro [77], blocked endothelial cell toxicity without reducing anti-tumor effect of Tf-CRM107 in vivo in nude mice bearing U251 human glioma xenografts [78]. Success of the Phase I trial prompted a Phase II clinical trial with Tf-CRM107 for the treatment of refractory and recurrent glioblastoma multiforme or anaplastic astrocytoma [76]. Forty-four patients were included in the trail. Thirty-one of those patients received two infusions of TfCRM107, while the remaining 13 patients only received one infusion. At the one-year evaluation, five of the 34 patients that received the two infusions showed complete responses, while seven showed partial responses, and nine demonstrated stable disease. The remaining 13 demonstrated progressive disease at the one-year evaluation. Fourteen percent of the 44 patients that received the Tf-CRM107 treatment showed cerebral edema that was managed by steroid or mannitol infusion. Seizures were observed in only three patients and were responsive to medical management. Phase III clinical trials to compare the antitumoral effect of Tf-CRM107 to those of standard chemotherapies against non-operable, progressive or recurrent glioblastoma multiforme are currently underway (National Cancer Institute’s Clinical trial identifier: NCT00087230).

Ribonuclease Tf, B3/25, and 42/6 have been conjugated to the bovine ribonuclease (RNAse) enzyme [79]. The RNAse immunotoxin showed dose dependent inhibition of protein synthesis and a decrease in clonogenic potential in the human K562 erythroleukemia cell line. Neither TfR antibody alone nor RNAse alone displayed any type of inhibitory effect. Excess Tf or inhibitors of RNAse blocked the effects of the conjugates confirming that the effects are dependent on targeting as well as the RNAse activity. Mixtures of Tf and RNAse (not conjugated) did not increase the inhibitory effects when compared to RNAse alone. This indicates that the two must be physically linked to have an inhibitory effect. The first chimeric antibody to target the TfR was developed in 1990 [80]. This chimeric antibody contains the human IgG1 Fc region and the variable region of the murine monoclonal anti-human TfR E6 antibody. Chimeric E6 was shown not to compete for Tf binding to the receptor. This chimeric antibody was subsequently used to develop fusion proteins that could deliver ribonuclease enzymes into malignant cells. Ribonuclease enzymes were chosen due to their decreased immunogenicity and non-specific cytotoxicities since they are normal blood proteins found in circulation. The first fusion protein developed using this chimeric antibody consisted of an anti-human TfR scFv fragment and a recombinant form of the human eosinophilderived neurotoxin (EDN), which is a part of the ribonuclease A superfamily and is not toxic by itself [81]. This scFvEDN fusion protein had only 6—13% ribonuclease activity compared to EDN alone, but demonstrated cytotoxic effects against the K562 and A431 human cell lines. The scFv of the chimeric E6 antibody was also genetically fused to angiogenin, an angiogenic member of the ribonuclease A family of enzymes [82]. The fusion protein was 6-

170 Table 4

T.R. Daniels et al. Delivery of high molecular weight compounds by human Tf or TfR antibodies

Conjugate

In vitro cytotoxicity

Tf conjugated Polymers/Polyplexes Tf-PEI-188Re

Tf-PEI-PEG-mouse TNFa gene

Tf conjugated Liposomes Mastoparan

ADR

Human erythroleukemia (K562) Human mammary metastatic cell line

Cisplatin

Endostatin gene a folate receptor antisense oligonucleotides

Various human breast cancer cell lines

Bcl-2 antisense oligonucleotides

Human erythroleukemia (K562)

Wild type p53 + radiotherapy

Wild type p53 +

AIPcS4 (photosensitizer) 10

B + thermal energy irradiation

Single chain anti-TfR conjugated liposomes 5E9 scFv-fluorescein labeled siRNA

Wild type p53

Tf modified viral vectors Adenovirus

Various human cancer cell lines

Human erythroleukemia (K562) Human ovarian cancer (OVCAR3)

Comments

References

Extensive tumor necrosis in human Burkitt’s lymphoma xenografts in nude mice; no leakage of nuclide to surrounding tissue. Inhibited tumor growth in 3 murine tumor models No systemic cytotoxicity observed.

[85]

Mastoparan accumulated in mouse colon carcinoma Colon26 tumors in mouse models. Cytotoxicity dependent upon ADR:lipid ratio. Prolonged the survival rates of nude mice bearing human gastric cancer MKN45 xenografts. Inhibition of angiogenesis and growth of mouse liver tumors. a folate receptor expression decreased by 60% Sensitized cells to ADR treatment. Increased sensitivity to daunorubicin 10-fold. Led to complete regression of human prostate cancer DU145 xenografts in nude mice. Decreased human prostate cancer PC3 xenografts. Reduced established human osteosarcoma HOSM-1 xenograft volume by 90% in nude mice. Photosensitizer accumulation in orthotopic rat bladder cancer model. Suppression of tumor growth and improvement of long term survival in mouse colon carcinoma tumor model.

[89,91]

siRNA accumulates in human pancreas and prostate cancer orthotopic xentografts in nude mice. 4- to 10-fold increase in transfection efficiency in nude mice bearing DU145 human prostate cancer xenografts. Systemic administration sensitized MDA-435/LCC6 (drug resistance) human breast cancer nude mice xenografts to docetaxel and significantly increased life span of the mice.

[86]

[92] [93]

[94] [95]

[96] [99,100]

[101]

[102] [103]

[105]

[106]

[107]

Increased transduction rates.

[108]

5-fold increase in reporter gene delivery.

[109]

Transferrin receptor

171

Table 4 (continued) Conjugate

In vitro cytotoxicity

Comments

References

Tf modified viral vectors Soluble coxsackie virus adenovirus receptor

Human colon carcinoma (Caco2)

Delivery of reporter gene.

[109]

Delivered to mice HT-29 xenografts. Uptake of targeted NP was 3-fold higher than nontargeted liposomes. IC50 was 5-fold lower than nontargeted NP or Paclitaxel alone. Complete tumor regression in nude mice bearing PC3 xenografts

[112] [113]

Nanoparticles (NP) Anti-c-myc DNAzymes Paclitaxel

Human prostate cancer (PC3)

fold less active in inhibiting protein synthesis compared to angiogenin alone. The scFv-angiogenin fusion protein was cytotoxic to the MDA-MB-231 (breast adenocarcinoma), HT29 (colon adenocarcinoma), and ACHN (renal carcinoma) human cell lines. F(ab’)2 fragments of the chimeric E6 antibody have been fused with human angiogenin as well [83]. This fusion protein inhibited growth of the K562 human cell line. The chimeric and parental E6 antibodies alone or used in combination (not genetically fused) with angiogenin demonstrated no growth inhibitory effects. A fusion protein consisting of angiogenin fused to the CH2 region of the E6 chimeric antibody also demonstrated cytotoxic effects against human cells lines, including MDAMB-231 (breast adenocarcinoma) and SF539 (glioma) [84]. Angiogenin remained fully active as part of this fusion protein.

Delivery of high molecular weight compounds Many tumor therapies seek to deliver therapeutic high molecular weight compounds including genes, which either restore the normal function of a defective gene and/or are capable of destroying the malignant cell. One of the major hurdles of these therapies is adequate delivery of the therapeutic agent into target cells. Genetically engineered viral vectors are highly efficient in delivery. However, viral vectors face potential problems including the induction the host immune response. There is a great need for viral vector alternative therapies that have a high efficiency of gene delivery. Recent advancements in the development of alternative therapies that target the TfR to deliver high molecular weight compounds into malignant cells are summarized in Table 4.

Polymers/Polyplexes Cationic polymers, such as polytheyleneimine (PEI), have been used as a gene delivery vector [85]. Human Burkitt’s lymphoma xenografts were established in nude mice prior to intratumoral injection of radioactive nuclide Rhenium-188 (188Re) labeled Tf-PEI conjugates. Increased retention of 188Re was observed in mice treated with the 188Re-Tf-PEI. Tumors in these mice demonstrated extensive necrosis without widespread leakage of the radionuclide to neighboring tissues.

Polyplexes consisting of plasmid DNA, PEI, polyethylene glycol (PEG) (to block non-specific interactions with blood components and other cells that have been previously reported with PEI polymers), and Tf (for tumor targeting) have also been evaluated as a delivery system. Systemic administration via intravenous injection of these polyplexes delivered the mouse tumor necrosis factor a (TNF a) gene and inhibited tumor growth in three murine neuroblastoma tumor models [86]. No sign of general toxicity or side effects were observed in any of these mice.

Tf conjugated liposomes Liposomes, synthetic vesicles enclosed by at least one phospholipid layer, are another therapeutic alternative to viral vectors. Liposomes can be used to deliver their contents (genes, chemotherapeutic drugs or other therapeutic agents) into tumor tissues. Liposomes are not immunogenic and can easily undergo size and lipid content modifications for targeting purposes [87,88]. The addition of PEG to the surface of liposomes extends the circulation half-life of the liposome by preventing opsonization of the liposomes by macrophages in the reticuloendothelial system [89]. A drawback of this liposome technology is the possible low transfection efficiency into target cells. This can be overcome by covalently coupling the liposomes to a molecule that targets tumors cells and mediates liposomal uptake. Human Tf has been previously used for this purpose because of its ability to deliver the liposomes into the intracellular compartment through receptor-mediated endocytosis [90]. Importantly, the coupling of PEG to Tf does not change its ability to interact with the TfR [89]. Mastoparan, a peptide derived from wasp venom, forms permeability transition pores in mitochondrial membranes leading the release of pro-apoptotic factors from this organelle. Mastoparan encapsulated by Tf-liposomes was delivered to human K562 cells and resulted in the release of cytochrome c from the mitochondria [91]. Systemic delivery of mastoparan-Tf-liposomes by intravenous injection enhanced uptake and accumulation of mastoparan in vivo in mouse colon carcinoma Colon26 bearing mice [89]. Tf-liposomes can deliver chemotherapeutic drugs into malignant cells as well. These targeted liposomes delivered

172 ADR to a human metastatic mammary cancer cell line and significantly enhanced cytotoxicity compared to non-targeted liposomes [92]. This tumoricidal effect was dependent on the ADR:lipid ratio. Higher ratios of ADR:lipids resulted in higher cytotoxicities. Tf-liposomes can also deliver drugs to tumor cells in vivo. Cisplatin was delivered to peritoneal xenografts of human gastric cancer MKN45 cells in nude mice [93]. Four days after tumor inoculation, mice received Tf-PEG liposomes via intaperitoneal injection. Uptake of TfPEG liposomes was significantly greater than non-targeted PEG or bare liposomes. Mice treated with Tf-PEG demonstrated prolonged survival rates compared to non-targeted PEG liposomes, bare liposomes, or cisplatin alone. Therapeutic genes or pieces of DNA can also be delivered to malignant cells via Tf targeted liposomes. Delivery of the anti-angiogenic endostatin gene by aerosol administration of Tf-liposomes to mouse liver tumor (isolated from Heps mouse) bearing mice inhibited angiogenesis and the growth of these tumors [94]. Tf liposomes were used to deliver antisense oligonucleotides to the alpha folate receptor to a variety of human breast cancer cell lines [95]. This receptor is overexpressed in a variety of malignancies and mediates the uptake of folic acid and reduced folates, which are essential for cell survival. In targeted cells, the expression of the alpha folate receptor was decreased by 60%. Treatment with the targeted liposomes carrying the alpha folate receptor oligonucleotides also sensitized human breast cancer cells to ADR. Tf-liposomes can also deliver Bcl-2 antisense oligonucleotides to human K562 cells [96]. Bcl-2 is an antiapoptotic protein frequently overexpressed in tumors and overexpression is associated with the resistance to chemotherapy, including resistance to daunorubicin [97]. Delivery of Bcl-2 antisense oligodeoxyribonucleotides resulted in a decrease in Bcl-2 expression and a 10-fold increase in K562 sensitivity to daunorubicin [96]. These effects were blocked by the addition of free Tf providing evidence that targeting via the TfR was essential. These studies provide strong evidence that therapies targeting the TfR can be used in combination with traditional chemotherapeutic drugs as beneficial treatment modalities for human malignancies. Liposomes targeting the TfR have also been extensively studied to deliver the tumor suppressor gene p53. p53 is a transcription factor that is activated upon DNA damage and has been termed the ‘‘guardian of the genome’’ [98]. Activation of p53 leads to cell cycle arrest, DNA repair, and in some cases cell death if the DNA damage is extensive. p53 is mutated in many types of tumors and this loss of function results in genomic instability and impaired apoptosis. The reintroduction of the wild type p53 gene into malignant cells has been a common goal for gene therapy. Tf-liposomes in combination with radiotherapy led to the complete regression of human prostate cancer DU145 xenografts in nude mice [99]. These liposomes also decreased the tumor volume of human prostate cancer PC3 xenografts [100]. The expression levels of p53 correlated with growth inhibition and increased survival of tumor bearing mice. Tf-liposomes that delivered the p53 gene also blocked the growth of established human osteosarcoma HOSM-1 xenografts in nude mice and decreased tumor volume to 1/10 that of control mice [101]. Alternative therapies can be used in combination with treatment of targeted liposomes. Photodynamic therapy is a

T.R. Daniels et al. treatment modality for superficial malignancies. This therapy consists of exposing a light source to photosensitizers. The products of this chemical reaction are reactive oxygen species that lead to the lethal oxidative damage of cellular components. The challenge in this type of therapy is targeting the photosensitizer to the malignant cells. Tf-liposomes have been used to deliver these photosensitizers to malignant cells [102]. In an in vivo orthotopic rat cancer model where AY-27 TCC bladder cancer cells are instilled in the bladder, intravesical injection (into the bladder) of the Tf-liposomes demonstrated increased accumulation of these photosensitizer molecules within tumor cells, while non-targeted liposomes did not accumulate in the animal. Injection of the photosensitizer alone demonstrated non-specific accumulation throughout the rat. Boron-neutron-capture therapy is another form of alternative anti-tumor therapy. The boron isotope (10B) interacts with thermal neutrons to produce cytotoxic byproducts. Delivery of 10B to Colon 26 mouse colon carcinoma tumors in mice via systemic intravenous injection of Tf-10B-liposomes increases 10B accumulation within the tumor for up to 72 h after delivery [103]. Irradiation with thermal energy suppressed tumor growth and improved longterm survival in Tf-10B-liposome treated mice.

Single chain antibody conjugated liposomes Immunoliposomes targeting the TfR have been constructed by covalent linkage of the single chain variable region of the 5E9 antibody (scFv anti-TfR) to liposomes [104,105]. These TfR targeted liposomes administered by intravenous injection efficiently delivered fluorescein-labeled small inhibitory RNA (siRNA) to human orthotopic xenografts, including Capan-I pancreas and PC3 prostate cancer xenografts, or lung metastases induced by intravenous injection of MDA435/LCC6 human breast cancer cells through the tail vein [105]. The scFv anti-TfR liposomes could efficiently target primary and metastatic cell lines of various cancer types and have also been used to deliver wild type p53 into nude mice bearing DU145 human prostate cancer xenografts [106]. Targeted liposomes showed a 4- to 10-fold increase in transfection efficiency and a scFv anti-TfR-liposome showed a 2-fold increase over Tf-liposomes in this model system. In the metastatic MDA-435/LCC6 (drug resistant) human breast cancer model, systemic administration of scFv anti-TfRliposomes containing the p53 gene sensitized the metastatic breast cancer cells to the anti-microtubule chemotherapeutic agent docetaxel (Taxotere) and significantly increased the life span of the mice [107]. These studies emphasize the advantage of combination therapy including immunoliposomes targeting the TfR to sensitize cancer cells to traditional chemotherapeutic drugs.

Modified viral vectors The enhanced delivery of therapeutic molecules into malignant cells by modified viral vectors has also been evaluated [108—110]. Cysteine residues were genetically added in the adenovirus capsid protein that allowed the coupling of this capsid protein to Tf [108]. Human K562 cells, which express low levels of the adenovirus receptor but high TfR levels, transduced with this Tf modified platform showed increased

Transferrin receptor transduction rates. A fiber-modified adenovirus carrying a Tf peptide on the capsid of the virus led to a 5-fold increase in the delivery of a reporter transgene in the OVCAR3 human ovarian cancer cell line [109]. A fusion protein that consisted of the soluble Coxsackie adenovirus receptor conjugated to Tf was constructed and used to deliver a non-replicative adenoviral vector encoding the luciferase reporter gene to Caco-2 human colon carcinoma cells [110]. Taken together these studies suggest that modified viral vectors targeting the TfR may enhance the delivery of therapeutic genes into tumor cells.

Nanoparticles Nanoparticles (NP) are defined as submicronic colloidal systems, may provide sustained drug effect and thus serve as a means to avoid systemic non-specific toxicities [111]. NP have been used to deliver DNAzymes, or short catalytic singlestranded DNA molecules to tumor cells as anti-tumor agents [112]. These particles consist of cyclodextrin-containing polycations that condense nucleic acids to form small NP. These NP are then stabilized using polyethylene glycol conjugated to transferrin for tumor targeting. These particles have been used to deliver anti-c-myc DNAzymes to mice bearing HT-29 human colon adenocarcinoma xenografts. The NP were successfully delivered and retained, while nontargeted NP were rapidly cleared from the body. Paclitaxel (TaxolR ) is a microtubule inhibitor that blocks depolymerization of microtubules and thus blocks cell division. Paclitaxel is commonly used to treat ovarian, breast, AIDS-related Kaposi’s sarcoma, lung cancer, and prostate cancer. While paclitaxel systemic administration causes sides affects such as hematopoietic, gut epithelial, and neurotoxic effects, a novel approach using NP conjugated to Tf to deliver Paclitaxel has recently been attempted and suggested for the treatment of localized prostate cancer [113]. Uptake of Paclitaxel-Tf-NP by the human prostate cancer cell line PC3 was 3-fold higher than Paclitaxel-NP alone and the IC50 for the Paclitaxel-Tf-NP was 5-fold lower than non-targeted NP or Paclitaxel alone. Nude mice injected intratumorally with Paclitaxel-Tf-NP demonstrated complete tumor regression and greater survival rate than NP or Paclitaxel alone against the human PC3 xenografts.

Conclusion The TfR is an attractive targeting molecule that can be used to treat a variety of malignancies. Targeting the TfR can occur via one of two ways: either through Tf itself, which targets both TfR1 and TfR2, or through the use of monoclonal antibodies specific for TfR1 and potentially specific for TfR2. Targeting the TfR has been shown to be effective in delivering therapeutic agents, including chemotherapeutic drugs, toxic proteins, and high molecular weight compounds into cells and causing cytotoxic effects including growth inhibition and/or induction of apoptosis in a variety of malignancies in vitro and in vivo including patients. The chimeric anti-TfR IgG3-Av antibody fusion protein developed by our laboratory is a unique molecule that exhibits both intrinsic cytotoxic activity with the ability to deliver a wide variety of biotinylated therapeutic agents into cancer cells. More advances in this area are expected to further improve the therapeutic potential of targeting the TfR.

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Acknowledgments This work was supported in part by grants K01 CA86915 and R01 CA107023 from NCI/NIH, the 2004 Brian D. Novis International Myeloma Foundation Senior Grant Award, and the 2003 Jonsson Cancer Center Foundation Interdisciplinary Grant ‘‘Targeted Therapy of Multiple Myeloma’’.

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