Enhanced HER-2 Directed Liposomal Therapeutics

Institution: California Pacific Medical Center Research Institute
Investigator(s): Daryl Drummond, Ph.D. - Daryl Drummond, Ph.D. -
Award Cycle: 2001 (Cycle VII) Grant #: 7KB-0066 Award: $332,938
Award Type: New Investigator Awards
Research Priorities
Detection, Prognosis and Treatment>Innovative Treatment Modalities: search for a cure

This is a collaboration with: 7KB-0066A -

Initial Award Abstract (2001)
Women suffer needlessly during treatments for breast cancer mainly because many drugs affect tissues other than the tumor due to their inability to be targeted specifically to the tumor. These problems are especially critical for chemotherapy. Our laboratory focuses on the development and engineering of lipid-based delivery systems, such as liposomes, for anti-cancer drugs and for gene therapy. In this strategy the drug or gene is contained within microscopic lipid droplets. We can increase the circulation time by reducing the tendency of the liposome to be taken up by the liver, and we can target the Iiposomes to breast cancer by attaching 'homing molecules' at the surface of the Iiposome. Even without these improvements there is a significant passive targeting of liposomes to breast cancers, because of the 'leaky' blood vessels supporting the tumor.

About 30% of breast cancer, especially the most aggressive type, have elevated amounts of a growth factor surface protein, called Her-2. Taking advantage of this, we have greatly increased liposome specificity by attaching human Her-2 single-chain antibodies to the surface of doxorubicin-containing liposomes. The results in animal tumor models experiments are encouraging. There is a 40-55% increase in efficacy compared to non-targeted liposomes or free doxorubicin. The National Cancer Institute is conducting preclinical toxicology experiments in monkeys and phase 1 clinical trials of this general liposome strategy.

There is still room for improvement. We propose to further optimize both drug release rates and tumor accumulation efficiencies for these drug-containing and antibody-targeted liposomes for an even greater therapeutic index. These enhanced properties will allow more drug to reach the cancer, while avoiding toxicity specific to long-circulating liposomes, such as Hand and Foot Syndrome. The main features of our approach include: (1) modifying the lipid composition of the liposome, (2) altering the method of drug entrapement inside liposomes, and (3) incorporating an acid-sensitive formulation. The purpose of making the liposome formulation acid-sensitive is to allow release of drug from the endosomal compartment within breast cancer cells following its internalization via the Her-2 antibody-receptor interaction. This will increase the amount of the active drug that is actually available to kill cancer cells, which should reduce the amount of total drug that needs to be administered. In the proposed studies we will measure how rapidly liposomal anti-cancer drugs arrive at the tumor as well as sites of potential toxicity. In addition, we will measure how fast the drug leaks from the liposomal carrier. Our aim is to search for the best strategy to deliver drugs to the breast cancer cell in a highly active form. For these experiments we use human breast cancers grown in mice.

Cancer researchers have been able to use toxic drugs to kill breast cancer cells in the laboratory for many decades, and novel therapies are continuously being developed. The use of liposomal drug carriers offers a potential solution to many vexing clinical issues that greatly diminishes the current effectiveness of chemotherapeutic drugs. Our goal is to increase survival and reduce the suffering of breast cancer patients by selectively and efficiently delivering the drug to the cancer and away from healthy tissues.

Final Report (2003)
Note: The PI has transferred the remaining 1 year of the award from the California Pacific Medical Center in San Francisco to Hermes Biosciences, Inc. in S. San Francisco.

Targeted delivery of anti-cancer drugs would serve to reduce the side-effects commonly observed with chemotherapy treatment strategies for breast cancer. The research of our laboratory has focused on the delivery of these drugs to cancer cells by way of “immunotargeted liposomes.” Thus, we encapsulate the chemotherapy agent, such as doxorubicin, within microscopic lipid particles, called liposomes, and direct them to the cancer by attaching an antibody fragment against the Her2/neu receptor. This results in the uptake of the drug-loaded liposomes by the cancer cells, which restricts the drug’s release and killing to the cancer cell itself. In our studies using human breast cancers grown in animal models we have found a dramatic increase (up to 70% “cures”) in efficacy compared to either non-targeted liposomes or free doxorubicin.

The aim of this research has been to optimize these immunotargeted drug delivery systems with respect to, (i) the rate at which the drug is released from the liposome, and (ii) measuring the efficiency of accumulation in tumors for an even greater therapeutic index. We have identified several new liposome formulations that have circulation lifetimes in the blood approximating those of “sterically stabilized” (high PEG) liposomes and retaining similar rates of tumor accumulation. These newly formulated liposomes resulted in considerable Her2-specific antitumor activity, although less cures than presently noted with the “highly pegylated” formulations at the same dose. Recently, we have determined this difference in efficacy was due in part to, (i) the immunodeficient nature of the animal, and (ii) the presence of estrogen for human breast tumor growth. Immunocompetent mice and those not receiving estrogen are currently being examined in an in vivo efficacy study using a protocol that we refer to as liposome "flooding" to further study this effect and to compare efficacy.

We have also prepared multiple liposomal formulations of a second, highly active anti-breast cancer agent, vinorelbine, with the aim of developing an additional therapeutic. We are continuing to test of the drug release rates in rats, and this work has given us greater insight into drug/tumor release rates that will guide future clinical trials.

In summary, the research supported by the CBCRP has allowed to refine our approach in terms of formulating and optimizing the liposomal drug agents that we anticipate taking to the clinic. In particular, understanding the relationship of drug formulation and delivery to the type of animal model and presence of estrogen has allowed to move past a technical hurdle and better plan for the final stages of our translational effort.

Final Report (2005)
Liposomes have been used successfully in the past to deliver anthracyclines, such as doxorubicin, for the treatment of cancer (Doxil, Alza Pharmaceuticals). However, the effectiveness of this drug delivery strategy has been difficult to replicate with other classes of anti-cancer drugs, including camptothecins and vinca alkaloids. The delivery problems are due to premature leakage of the drug from the liposomal carrier while in the circulation, thus depriving the carrier of the ultimate promise of site-specific drug delivery. Highly stable nanoliposome constructs have been developed with the aid of this grant that utilize novel trapping agents to form stable drug complexes in the interior of the particle, significantly retarding drug release until after the drug-carrier combination has had time to accumulate in the tumor. The anticancer vinca alkaloid, vinorelbine, is used to demonstrate the effectiveness of the technology and the result is a markedly active drug when compared to the un-encapsulated drug. This is in sharp contrast to other nanoparticle vinca alkaloid drugs, which leak rapidly from their carriers. We believe this technology is broadly applicable to a wide range of anticancer drugs and will improve the therapeutic index of breast cancer therapeutics significantly, allowing for increased survival and quality of life for patients with breast cancer. An antibody-targeted version of this therapeutic agent using anti-HER2 and anti-EGFR antibody fragments has demonstrated even greater efficacy in animal tumor models and improves the potential for disease-specific anti-tumor activity and reduced toxicity.

In addition, we have looked at how modifications of the liposomal nanoparticle can affect the pharmacokinetics and accumulation of liposomal and immunoliposomal drugs at the site of the tumor. We have found that small amounts of aggregation-reducing lipids, including PEG-DSPE and glutaryl-DSPE are able to prolong circulation lifetimes of doxorubicin-loaded liposomes. However, neither of these stabilizing agents were as effective as high concentrations of pegylated phospholipids in improving antitumor efficacy of a HER2-targeted immunoliposomal doxorabicin. In addition, the presence of high concentrations of PEG on the surface of the nanoparticle allowed for a greater number of modifications to the liposomal carrier, or its mode of delivery, to be made with minimal adverse effects on its drug delivery potential. Pegylation did not adversely affect binding of the targeted liposomal doxorubicin to its target HER2-epitope or HER2 specific cytotoxicity.

In summary, the research supported by the CBCRP has provided a better understanding of the basic criteria important for specific drug delivery using immunoliposomes. It has also helped us expand the number of therapeutic agents that can be effectively delivered using immunoliposomes and provided new hope for developing highly effective treatment options for women suffering with breast cancer.

Symposium Abstract (2003)
Site specific delivery of anti-cancer therapeutics is paramount for both reducing nonspecific toxicities and increasing efficacy of conventional forms of breast cancer chemotherapy. Due to their small size and nonspecific mechanisms of action, most conventional chemotherapy results in significant toxicities that limit the effectiveness of treatment and reduce the overall quality of life for cancer patients. Our laboratory focuses on the development of liposomal delivery systems, targeted specifically to cancer cells that express high levels of growth factor receptors on their cell surface. The attachment of human anti-HER2 single chain antibodies to the surface of liposomes results in their internalization by certain very aggressive breast cancer cells and a “trojan horse” effect, where the drug is delivered to the inside of the cancer cell using receptors that normally bind growth factors the cancer requires for survival. The result is a dramatic increase in efficacy compared to nontargeted liposomes or free doxoru-bicin in a human breast tumor model, with 40-55 % cures noted.

The aim of this project has been to optimize these immunotargeted drug delivery systems with respect to the rate at which the drug is released from the liposome and efficiency of accumulation in tumors for an even greater therapeutic index. We have identified several formulations that have circulation lifetimes in the blood approximating those of steri-cally stabilized liposomes and similar rates of tumor accumulation. These liposomes resulted in considerable HER2-specific antitumor activity, although less cures than presently noted with the highly pegylated formulation at the same dose.

Concentrating on a second highly active anticancer agent, we have prepared multiple formulations of liposomal vinorelbine in hopes of developing a second targeted liposomal therapeutic for the treatment of breast cancer with a release rate from the carrier that would approximate that of delivery to the tumor. Testing of the drug release rates from the liposomal carrier in vivo in rats has shown that we indeed have prepared formulations with a wide range of drug release rates. Examination of activity in cell culture indicated significant anticancer activity for all formulations, but a degree of target specificity that varied depending on the formulation. Several of these formulations are currently being tested in antitumor efficacy studies in a HER2-overexpressing breast tumor model. The hope we hold is that these optimized and targeted delivery systems will reduce the suffering of women with breast cancer and increase their survival.

Symposium Abstract (2005)
Targeted delivery of anti-cancer drugs would serve to reduce the side-effects commonly observed with chemotherapy treatment strategies for breast cancer. The research of our laboratory has focused on the delivery of these drugs to cancer cells by way of “immunotargeted liposomes.” Following the successful clinical development of iposomal anthracyclines (doxorubicin, daunorubicin), vinca alkaloids are the next most likely candidates to join the list of clinically approved liposomal anticancer agents. However, in contrast to anthracyclines, liposome-encapsulated vincas suffer from increased drug leakage rates in vivo due to the inherently higher hydrophilicity of these drugs. We have developed a novel liposome drug loading strategy employing transmembrane gradients of the salts formed by polyalkyl-substituted amines with highly charged anions that allowed for improved loading capacity, loading efficiency, and greatly improved in vivo drug encapsulation stability of anticancer vinca alkaloids. This strategy was applied to vinorelbine, an attractive candidate for liposomal delivery due to its favorable toxicity profile, positive clinical experience in breast and lung carcinomas, and a chemical structure amenable to active loading into liposomes via transmembrane gradient approach. A series of liposomal vinorelbine formulations using various drug loads and intraliposomal drug-stabilizing anions were prepared, with drug-to-lipid ratios of up to 0.5 g/mmol phospholipid, and encapsulation efficiency over 90%. In vivo drug release rates in the blood of rats varies from drug release half-life of 1.8 hours to over 24 hours. These formulations showed prominent anti-tumor activity in several human and syngeneic tumor models in mice. The most active of these formulations is currently in Phase I clinical trials.

The suitability of these novel vinorelbine liposome formulations for antibody-directed targeting (immunotargeting) was also studied. HER2- or EGFR- targeted immunoliposomal vinorelbines formulations were prepared from the amphiphilic PEG-DSPE conjugated of anti-HER2 scFv or anti-EGFR Fab' and preformed vinorelbine liposomes using a micellar insertion method. The immunoliposomal vinorelbines showed target-specific cytototoxicity in cell cultures and good in vivo stability. Antitumor efficacy of these formulations tested in HER2-overexpressing breast tumor and EGFR-overexpressing tumor xenografts in nude mice was target specific and dependent on the rate of drug release from the liposome, with more stable constructs showing greater efficacy. Thus, better anticancer properties of liposomally formulated vinca alkaloids were achieved through improved drug encapsulation stability and cancer cell-specific targeting. These novel and highly active molecularly targeted therapeutics offer a promising treatment potential and improved toxicity profile for women with breast cancer.

Her2- and EGFR-targeted liposomal epirubicin.
Periodical:American Association for Cancer Research
Index Medicus: AACR
Authors: Guo et al.
Yr: 2003 Vol: March Nbr: Abs: Pg:

Immunoliposomes target EGFR-overexpressing brand and other tumors for efficient drug delivery and enhanced efficacy.
Periodical:American Society for Clinical Oncology
Index Medicus: Am Soc Clin Oncol
Authors: Mamot et al.
Yr: 2003 Vol: Nbr: Abs: Pg:

Membrane determinants of in vivo drug clearance and anti-tumor efficacy with liposomal duxorubicin.
Periodical:Am. Assoc. Pharm. Sci.
Index Medicus: Am Assoc Pharm Sci
Authors: Noble et al.
Yr: 2003 Vol: Nbr: Abs: Pg:

Membrane determinants of long circulation and in vivo liposomal drug efficacy.
Periodical:American Association for Cancer Research
Index Medicus: AACR
Authors: Noble et al.
Yr: 2003 Vol: March Nbr: Abs: Pg:

Epidermal Growth Factor Receptor (EGFR)-targeted Immunoliposomes Mediate Specific and Efficient Drug Delivery to EGFR- and EGFRvIII-overexpressing Tumor Cells.
Periodical:Cancer Research
Index Medicus: Cancer Res
Authors: Mamot C, Drummond DC, Greiser U, Hong K, Kirpotin DB, Marks JD, Park JW
Yr: 2003 Vol: 63 Nbr: 12 Abs: Pg:3154-3161