WO2000064476A2 - Immuno-adjuvant pdt treatment of metastatic tumors - Google Patents

Abstract

Immuno-adjuvant photodynamic therapy to treat and prevent metastatic cancer is effected using photosensitizers in combination with immuno-adjuvants to destroy metastatic tumor cells.

Description

IMMUNO-ADJUNANT PDT TREATMENT OF METASTATIC TUMORS

Field of the Invention

The invention relates to the use of photodynamic therapy (PDT) treatment in combination with immuno-adjuvants to treat metastatic tumors. The PDT may be conducted with any photosensitizer, but combinations comprising a benzoporphyrin derivative (BPD) are preferred for such PDT treatment.

Description of the Related Art

This invention relates to metastatic cancer. The metastatic process, which results in the growth of secondary tumors at sites distal to the primary tumor, is the cause of death in most cancers (Poste and Fidler, 1980). Although most patients with newly diagnosed solid tumors are free of detectable metastases, and about half of those patients can be cured of their disease by local cancer treatment, the remaining patients have clinically occult micrometastases that will become evident with time. Thus, at the time of primary tumor treatment, the total percentage of patients with either detectable metastases or microscopic disseminated disease is 60% (Liotta and Stetler-Stevenson, 1989).

The brain is the most favored site for metastatic spread, occurring in 25% to 30% of all cancer patients: the most frequent primary cancers, lung cancer, breast cancer and melanoma, are associated with high incidence of brain metastases (Wright and Delaney, 1989). The lung is the second most common site of metastatic spread and pulmonary metastases most frequently originate from bone and soft-tissue sarcomas (Roth, 1989). Liver metastases commonly result from gastrointestinal tract tumors (Sugarbaker and Kemeny, 1989) and bone metastases from breast, lung and kidney primary tumors (Malawer and Delaney, 1989).

Management of a significant number of cancer cases, therefore, depends upon treating multiple tumors, traditionally through the use of surgery, radiation therapy, chemotherapy, or adjuvant therapies consisting of combinations of the three modalities.

Observations relating to tumor immunity have provided a focal point for the development of possible tumor therapy. Prehn and Main showed in 1957 that chemically induced tumors of mice were antigenic. There has been controversy concerning the relevance of chemically induced tumors, which are generally immunogenic, compared with spontaneously arising tumors in mice and human tumors which are not (Hewitt, 1979; Hewitt et al., 1976).

The issue was addressed by Boon et al. who showed that mutagenized antigenic variants of non-immunogenic tumors could generate immunological protection in mice against the parent tumor; that is, the mutagenized and parent tumors shared antigens (Boon et al., 1994). The results suggested that spontaneous experimental tumors and human tumors were antigenic and could be made immunogenic through the appropriate augmentation of the immune system (Boon et al, 1994). Subsequent studies confirmed that the immune system could be made to recognize weakly immunogenic tumors by transforming tumor cells with genes for the expression of cytokines, co-stimulatory molecules, or MHC molecules (Gajewski et al., 1995; Pardoll, 1993).

Also, in vitro culture of tumor-infiltrating lymphocytes from tumor-bearing mice and cancer patients with cytokines and irradiated tumor cells, and re-infusion of the activated lymphocytes can result in tumor regression (Burger et al., 1996; Schultze et al., 1997). Finally, tumor antigens recognized by the cells of the immune system have been identified in both animal models and human tumors (Jaffee and Pardoll, 1996). Tumor antigens recognized by T lymphocytes in human melanomas are the most fully characterized set of tumor antigens and may be non-mutated, widely distributed molecules, unique and mutated proteins, or normal proteins that are overexpressed in tumors (Robbins and Kawakami, 1996).

One result from the observations concerning tumor immunity is cancer immunotherapy. For centuries it has been observed that many types of diseases, including cancer, can be improved or even cured following attacks of erysipelas, an acute skin infection. In 1909 William Coley reported several positive results following deliberate infection of cancer patients with bacteria in order to induce erysipelas. Although the contemporary theory explained tumor improvements or cures as the result of toxic products released during the bacterial infection, Coley's approach to cancer treatment may be regarded as the first instance of "biotherapy" (the original term) or cancer immunotherapy.

Immunotherapy of cancer, in which the immune system is modulated through the use of specific and non-specific tumor vaccines, bioactive molecules such as cytokines, or adoptive transfer of activated lymphocytes is one of the most appealing approaches to the treatment of metastatic cancers . The therapy is based on the concept that the patient's immunological tolerance of their cancer can be broken so that the cancer is recognized as foreign by the patient's immune system (Gore and Riches, 1996).

Another tumor treatment method is photodyanamic therapy (PDT). PDT is based upon dye-sensitized photooxidation of diseased tissue and was originally developed as a treatment modality for solid tumors (Dougherty et al., 1975). Singlet oxygen (O,) is generated, without radical formation, through energy transfer processes from light- activated photosensitizer molecules in the "type II mechanism", and it is widely accepted that 'O is responsible for the primary photodynamic effect in vivo (Weishaupt et al., 1976). Membrane damage brought about by O_:-mediated lipid peroxidation leading to loss of cell integrity is thought to be the primary mode of cell killing by PDT (Henderson and Dougherty, 1992), although metabohcally regulated processes may also be involved in PDT-induced damage and cell death (Granville et al.. 1998; Tao et al., 1996).

Photosensitizers are usually delivered intravenously and selective destruction of tumor tissue is based upon preferential uptake of the drug by neoplastic tissue and localized exposure of the tumor to the wavelength of light best suited to tissue penetration and photosensitizer activation. Necrosis of tumor tissue is a result of the direct effects of 'O₂ on tumor cells, and also from the anoxic conditions that develop in the tumor following disruption of tumor vasculature by PDT (Henderson et al., 1985).

Following PDT, immune responses are initiated with the rapid induction of an inflammatory reaction (Henderson and Dougherty, 1992; Ochsner, 1997) involving the release of cytokines (Evans et al., 1990; Gollnick et al., 1997; Nseyo et al., 1989), eicosanoids (Fingar et al., 1991 ; Henderson and Donovan, 1989), and clotting factors (Fingar et al., 1990; Foster et al., 1991), and progresses to the activation of immune cells (Qin et al., 1993; Yamamoto et al., 1992; Yamamoto et al., 1994) and infiltration of immune cells into PDT-treated tissue (Korbelik et al., 1996). For example, tumor cells pre-treated with PDT in vitro were sensitised to macrophage-mediated lysis (Korbelik et al. 1994) and at low photosensitizer levels, PDT activated macrophage phagocytic activity (Yamamoto et al. 1994). Photofrin®-based PDT stimulated the release of the immunomodulatory molecules prostaglandin-E2 (Henderson et al. 1989) and tumour necrosis factor-α (TNF-α) (Evans et al. 1990) from murine macrophages. Photo frin® and light treatment induced the expression of interleukin (IL) IL-6 in HeLa cells (Kick et al. 1995) and within mouse tumours (Gollnick et al. 1997). A massive and rapid influx of granulocytes and macrophages has been described for murine tumours treated with Photo frin® and light [Golnick et al. 1997; Korbelik 1996; Krosl et al. 1995)]. PDT has been described as inducing tumor immunity (Korbelik 1996) which may be augmented by the localised administration of an adjuvant at the time of photo-irradiation (Korbelik et al. 1998). Moreover, granulocyte-macrophage colony stimulating factor (GM-CSF) administered in three doses at two-day intervals, commencing 48 hours before light- irradiation, improved the curative effect of Photofrin® and verteporfin-mediated PDT against mouse tumours (Krosl et al. 1996).

PDT has also been shown to enhance both phagocytosis and tumor cytotoxicity when normal mouse peritoneal macrophages were treated in vitro (Yamamoto et al., 1992; Yamamoto et al., 1994) and similar treatments caused the secretion of tumor necrosis factor (TNF) (Evans et al., 1990). In the clinical setting, treating bladder cancer with PDT resulted in detectable levels of interleukin (IL-1) and TNF-α in the urine of patients within 3 hours of treatment and IL-2 within 24 h in a profile that resembled treatment of bladder cancer with Bacille Calmette Guerin (BCG). In BCG therapy, elevated cytokine levels were associated with improvement (Evans et al., 1990).

The role of the host immune system in PDT-mediated tumor eradication has recently been examined by Korbelik et al. by comparing the response to PDT of a solid tumor grown in immunocompetent or immunodeficient mice. PDT cured all normal mice; however, using the same treatment protocol with nude mice (which have a congenital absence of the thymus, resulting in reduced numbers of T cells but normal levels of B and NK cells) or scid mice (which are unable to complete V(D)J recombinations during T and B cell development and have no mature T and B cells), the initial tumor ablation following PDT was followed by regrowth of all of the tumors Transfemng splenic T cells to scid mice or reconstituting lethally irradiated scid mice with normal mouse bone marrow pπor to PDT resulted in delayed regrowth or tumor cure (Korbelik et al , 1996)

The same group observed a 200-fold increase in the number of tumor-associated neutrophils within minutes of sub-optimal photodynamic treatment and a drop in neutrophil content to near control levels at 2 hours after light treatment (Krosl ei al , 1995) Infiltrating mast cell numbers also increased within 5 mm of light treatment and the higher level of mast cells was maintained for 4 hours after PDT The numbers of mast cells were, however, several logs lower than the numbers of neutrophils Approximately 10% of the total number of cells in the tumor at 2 hours after PDT were characteπzed as monocytes that had invaded the tumor from the circulation

Also, there was a large population (20% of total cells) of tumor-associated macrophages in untreated tumors Resident macrophages were equally sensitive to PDT killing as malignant cells but following PDT, tumor associated macrophages were shown to be almost 5 times more cytotoxic against tumor target cells in vitro, compared with macrophages isolated from untreated tumors

Another means of stimulating the host immune response is by the use of adjuv ants Any matenal that increases the immune response towards an antigen is referred to as an adjuvant (see Appendix A) and while they have been used for at least 70 years in the production of traditional vaccines designed to prevent infectious diseases, adjuvants are also being developed for use in cancer vaccines Adjuvants are able to augment immune responses through several mechanisms including 1 ) causing depot formation at the site of inoculation, 2) acting as delivery vehicles which may target antigens to cells of the immune system, 3) acting as immune system stimulators

Many of the adjuvant preparations function via several of these mechanisms The ideal adjuvant would have safe local and systemic reactions (which would preclude general toxicity, autoimmune and hypersensitivity reactions, and carcinogenicity) be chemically defined so consistent manufacture is possible, would enhance protective (or in the case of cancer vaccines, therapeutic) immunity towards weak antigens, and would be biodegradable (Audibert and Lise, 1993, Cox and Coulter, 1997, Gupta and Siber, 1995)

The prototypical adjuvant, which is also the most potent, is Freund's Complete Adjuvant (CFA) developed in 1937 by Jules Freund CFA consists of a preparation of killed Mycobacterium tuberculosis dispersed in mineral oil When emulsified with water soluble antigens, the vaccine stimulates both humoral (antibody-mediated) and cell- mediated immunity towards the antigens The use of this adjuvant may result in seπous side effects including organ injury via granuloma formation and autoimmune disease, and its use is restπcted even in experimental animals Incomplete Freund's Adjuvant (IFA), which lacks the mycobacteπal component of CFA, is less toxic but does not enhance cell- mediated immunity Nonetheless, IFA is currently undergoing clinical tπals in cancer vaccine formulations (for example NCI-T97-01 10, NCI-98-C-0142, NCI-H98-0010, NCI- T96-0033)

New adjuvants, such as the Ribi Adjuvant System (RAS) have been designed to substitute highly puπfied bacterial components for M tuberculosis in order to maintain the immune stimulatory properties of CFA without the side effects A vaπation of RAS, Detox™ adjuvant, is currently in clinical tπals as a component of cancer vaccines (NCI- V98-1489, NCI-96-C-0139) Others, such as Hunter's TiterMax, which is has not been approved for clinical use but has been extensively characteπzed in animal systems, use completely synthetic compounds

There have been previous attempts to combine immuno-adjuvants and PDT Myers et al injected formalin killed bacteπa, Corynebacterium parvum, intralesionaily in expeπmental tumors 24 hours pπor to PDT in the first reported case of immuno-adjuvant PDT The therapy improved the efficacy of hematoporphyπn deπvative (Hpd)-sensιtιzed PDT as measured by reduction tumor volume and prolongation of survival (Myers et al , 1989)

Using intralesional BCG, Cho et al followed a similar protocol as Myers et al to use PDT on a muπne transitional cell carcinoma model (Cho et al , 1992)

Korbehk's group reported results using immuno-adjuvant PDT in 1993 (Korbelik et al , 1993) Initially, the group administered the immunostimulant schizophyllan (SPG), a glucan derived from Schizophyllum communae, in a series of intramuscular injections into the hind leg of mice bearing a squamous cell carcinoma solid tumor grown mtradermally over the sacral region of the back. Photo frin-based PDT was administered either 48 hours after the last SPG treatment or 24 hours before the first SPG injection. SPG therapy before PDT enhanced the effect of PDT on tumor cure whereas immunotherapy after PDT had no effect (Krosl and Korbelik, 1994).

Another study found that administering the macrophage activating factor vitamin D₃ binding protein macrophage activating factor (DBPM.AF) intraperitoneally and peritumorally in a series starting immediately following Photofrin-sensitized PDT enhanced the PDT effect on tumor cures (Korbelik et al., 1997). Later, the group examined the use of BCG and a purified and deproteinized preparation of the mycobacterium cell wall extract (MCWE) that is distributed by Bioniche Inc. (London, Ont. Can.) as Regressin, combined with PDT sensitized with Photofrin, Verteporfin, zinc(II)-phthalocyanine (ZnPC), and /netαtetrahydroxyphenyl-chlorin (mThPC). A single injection of either MCWE or BCG directly beneath the tumor mass and immediately following PDT resulted in enhanced tumor cure rates (Korbelik and Cecic, 1998).

Nordquist et al. (U.S. Patent 5,747,475) disclose that the treatment of primary tumors in a rat model with indocyanine green (ICG) as chromophore and glycated chitosan as an immuno-adjuvant in photothermal therapy. This treatment resulted in some instances of reducing both primary and metastatic tumors as well as some instances of preventing the occurrence of metastatic tumors (see Figures 1 and 2 for effects against primary tumors; Figure 4 for effects against metastatic tumors; and Figure 5 for prevention of metastatic tumors).

Chen et al. combined glycated chitosan gel (GCG) prepared from crabshell chitin, with indocyanine green (ICG), injected ICG-GCG intratumorally and activated the ICG with thermal laser illumination in a rat metastatic tumor model. The treatment resulted in: a) no tumor response followed by death at 30 days post-treatment; b) reduced tumor burden and extended survival times to 45 days; and c) reduced tumor burden but continued growth of the treated tumor, followed by reduction of both the treated primary and untreated metastasis. Some of the animals in the last group were cured of their tumors and rejected a subsequent challenge with the same tumor cells, indicating that the animals had developed tumor immunity and immunological memory (Chen et al., 1997).

In the above instances, the processes were directed toward discrete or defined, localized tumors. Also, both Nordquist et al. and Chen et al. utilized photothermal mediated cell destruction as opposed to the photochemical mediated PDT discussed below, which does not cause any appreciable heating of the target tissue. Thus experimental combinations of immuno-adjuvants and PDT were attempted with little predictability as to actual efficacy and general application. Even the patent by Nordquist et al. only discloses the results from limited application of this concept with a single combination of one immuno-adjuvant (glycated chitosan) and one chromophore (ICG).

Given that the immune system plays an essential role in tumor destruction and the cytotoxic action of PDT, the present invention relates to a new therapeutic regime combining immunotherapy and PDT for the treatment and prevention of metastatic cancer.

Summary of the Invention

The invention is directed to the use of photodynamic therapy (PDT) in combination with immuno-adjuvants to treat, prevent, or inhibit the development of any tumor, especially metastatic tumors. In particular, photodynamic methods employing a photosensitizer, such as a benzoporphyrin derivative (BPD), a green porphyrin, are used in combination with an immuno-adjuvant against metastatic cancer after diagnosis. Additional applications of the combination are after any primary treatment method against a diagnosed tumor to prevent the onset of as yet undetected dissemination of metastatic tumors or to treat such tumors after their appearance. The instant methods offer the benefit of efficacy against non-localized metastatic tumors either before or after their detection.

Accordingly, in one aspect, the invention is directed to a method to treat metastatic tumors, which method comprises administering to a subject with such tumors an effective amount of a photosensitizer, such as a BPD, in combination with an immuno-adjuvant and irradiating the subject with light absorbed by the photosensitizer. Such methods may be employed against metastatic tumors upon initial diagnosis of cancer in a subject or against metastatic tumors that arise after previous tumor or cancer therapy in the subject. In another aspect, the invention is directed to a method to prevent or inhibit the development of metastatic tumors by the steps of administering to a subject previously having undergone cancer or tumor therapy, an effective amount of a photosensitizer, such as a BPD, in combination with an immuno-adjuvant and irradiating the subject with light absorbed by the photosensitizer. Such methods are employed even before the detection of metastasis and as such prevent, or reduce the occurrence of, metastatic tumors. The methods of the present invention specifically are contemplated for the administration of BPDs, such as those selected from the group consisting of BPD-DA, BPD-DB, BPD-MA (including BPD-MA-A also known as verteporfin) and BPD-MB (where BPDs are as presented in U.S. Patent 5, 171,749. which is hereby incorporated by reference as if fully set forth) as well as the derivatives of these compounds. Particularly preferred BPDs include BPD-MA, EA6 (including A-EA6. also known as QLT 0074) and B3, where EA6 (as set forth in U.S. Patent 5,929,105, which is hereby incorporated by reference as if fully set forth) and B3 (as set forth in U.S. Patent 5,990,149, which is hereby incorporated by reference as if fully set forth) have the following structures.

BPD-MA

B-EA6

A-B3

B-B3

The methods of the present invention may be practiced with any immuno-adjuvant or combination of immunoadjuvants, including those set forth in Appendix A. Particularly preferred immuno-adjuvants are those of microbial or crustacean (chitosan) derived products. These include the Ribi Adjuvant System, Detox™, glycated chitosan, and TiterMax™. The Ribi Adjuvant System and its components are described in issued US Patents 4,436,727 and 4,866,034. Preferably, the immuno-adjuvant comprises a mycobacterial cell wall skeleton component (described in US patent 4,436,727) and a component derived from lipid A of a bacterial lipopolysaccharide. Most preferably, the lipid A component is de-3-O-acylated monophosphoryl lipid A (described in US Patent 4,912,094. Additional adjuvants for use with the present invention include CFA. BCG, chitosan, and IFA. Delivery of the immuno-adjuvant may be systemic or localized.

Regarding compositions, the present invention includes pharmaceutical compositions to treat or prevent or inhibit the development of metastatic tumors, such compositions containing an amount of a photosensitizer in combination with an immuno- adjuvant effective to treat, prevent or inhibit development of metastatic tumors when administered to a subject followed by irradiation with light absorbed by the photosensitizer, and a pharmaceutically acceptable carrier or excipient. Compositions individually containing the photosensitizer and immuno-adjuvant for use together as needed are also encompassed.

Brief Description of the Drawings

The present invention will be more clearly understood by referring to the following drawings, in which:

Figure 1 shows biopsies containing experimental metastases in lungs of animals treated with immuno-adjuvant PDT, PDT only, and untreated controls.

Figure 2 shows in vitro lymphocyte proliferation in the presence of tumor antigens. See Example 4 below. The lymph nodes of mice bearing the Lewis Lung Carcinoma (LLC) cells were removed 7-10 days following treatment with PDT or PDV. Single cell suspensions of lymphocytes were cultured in the presence of LLC and accessory cells and incubated for 5 days after which proliferation was assessed using MTS. Detailed Descπption of the Invention

The present invention is directed to a procedure in which immuno-adjuvant photodynamic therapy (PDT) targets tumors, especially metastatic tumors, in some instances even before they are detectable Thus the invention may be applied against metastatic tumors including, but not limited to, those that oπgmate and/or result in melanoma, lung cancer, breast cancer, colon cancer, and prostate cancer The invention may also be used in cases of lymphoid tumors that form masses For treating metastatic tumors that have been newly diagnosed, this treatment may be utilized as a primary therapy against the tumors For preventing or inhibiting the development of metastatic tumors, this treatment may be used as additional or follow-up therapy after pπmary therapy against a diagnosed tumor

Thus following identification of metastatic tumors in a subject, an appropπate photosensitizing compound, preferably BPD-MA, EA6 or B3, will be administered to the subject in combination with an immuno-adjuvant The order of administration of photosensitizer and immuno-adjuvant may vary, with light lπadiation following administration of the photosensitizer The immuno-adjuvant may be administered immediately after light irradiation Simultaneous activation of the immune system by the immuno-adjuvant and PDT mediated damage to tumor cells, or initiation of immune reactions, may increase the effectiveness of treatment

After administration, the photosensitizer will localize in tumor cells for photoactivation while the immuno-adjuvant proceeds to activate/potentiate the immune response Light of appropπate frequency and intensity will be applied using an appropπate light source, thereby activating the photosensitizer to destroy tumor cells and initiate immune responses, possibly by the rapid induction of an inflammatory reaction

The formulations and methods of the present invention generally relate to admmistenng a photosensitizer, including pro-drugs such as 5-amιnolevulιmc acid, porphyπns and porphynn deπvatives e g chloπns, bacteπochloπns, isobacteπochloπns phthalocvanine and naphthalocyanines and other tetra- and poly-macrocyclic compounds, and related compounds (e.g pyropheophorbides) and metal complexes (such as, but not limited by, tin, aluminum, zmc, lutetium) to a subject undergoing the immuno-adjuvant PDT Examples of photosensitizers useful m the invention include, but are not limited to, the green porphyπns disclosed in a seπes of patents including US Patents 5,283,255, 4,920,143, 4,883,790, 5,095,030, and 5,171,749, and green porphynn derivatives, discussed in US Patents 5,880,145 and 5,990,149, all of which are hereby incorporated by reference as if fully set forth

Green porphyπns are in the class of compounds called benzoporphyπn derivativ es (BPD) A BPD is a synthetic chloπn- ke porphynn with vaπous structural analogues, as shown in U S Patent 5,171 ,749 Preferably, the BPD is a benzoporphyπn derivative di- acid or mono-acid πng A (BPD-DA or BPD-MA, also known as verteporfm), which absorbs light at about 692 nm wa elength with improved tissue penetration properties

BPD-MA, for example, is lipophihc, a potent photosensitizer, and it also appears to be phototoxic to neovascular tissues, tumors and remnant lens epithelial cells Because of its pharmokinetics, BPD-MA may be the best candidate for use in the instant invention, but other BPDs such as EA6 and B3 or other deπvatives may be used instead Other photosensitizers, such as phthalocyamnes, could be used m high concentrations sufficient to offset their relatively slower uptake An optimal BPD for immuno-adjuv ant PDT treatment or prevention of metastatic tumors should be rapidly taken up by tumor cells and should be capable of initiating an immune response upon irradiation with light to act in concert with the immuno-adjuvant

Other non-hmitmg examples of photosensitizers which may be useful in the invention are photosensitizing Diels-Alder porphynes deπvatives, descπbed in US Patent 5,308,608, porphyπn-hke compounds, descπbed in US Patents 5,405,957, 5,512675, and 5,726,304, bacteπochlorophyll-A deπvatives descπbed in US Patents 5,171,741 and 5,173,504, chloπns, isobacteπochloπns and bacteπochloπns, as descπbed in US Patent 5,831,088, meso-monoiodo-substituted and meso substituted tπpyrrane, descπbed in US Patent 5,831,088, polypyrrohc macrocycles from meso-substituted tnpyrrane compounds, descπbed in US Patents 5,703,230, 5,883,246, and 5,919,923, and ethylene glycol esters, descπbed in US Patent 5,929,105 All of the patents cited in this paragraph are hereby incorporated by reference as if fully set forth Generally any hydrophobic or hydrophi c photosensitizers, which absorb in the ultra-violet, visible and infra-red spectroscopic ranges would be useful for practicing this invention.

The preferred compounds of the present invention are the photosensitive compounds including naturally occurring or synthetic porphyrins, pyrroles, chlorins, tetrahydrochlorins, pyropheophorphides, purpurins, porphycenes, phenothiaziniums, pheophorbides, bacteriochlorins, isobacteriochlorins, phthalocyanines, napthalocyanines, and expanded pyrrole-based macrocyclic systems such as, sapphyrins and texaphyrins, and derivatives thereof. Other photosensitizers for use in the present invention are described in Redmond et al, Photochemistry and Photobiology, 70(4):391-475 (1999), which is hereby incorporated by reference in its entirety as if fully set forth. Preferably, the photosensitizer is not Photofrin™ (porfimer sodium).

A particularly preferred formulation according to the present invention will satisfy the following general criteria. First, an immuno-adjuvant capable of activating or potentiating the immune response is utilized. Second, a photosensitizer capable of rapid entry into the target tumor cells is used. Third, iπadiation with light results in cytotoxicity to target tumor cells. This then results in the generation of immune responses. These criteria do not necessarily reflect a temporal sequence of events.

In one embodiment, the methods of the invention are used against metastatic tumors after initial diagnosis. In another embodiment, the methods of the invention follow removal or eradication of a solid tumor by conventional treatments such as surgery, radiation, chemotherapy or PDT, including immuno-adjuvant PDT. The latter embodiment may be used to prevent or inhibit the development of, metastatic tumors.

In practice of the invention, the immuno-adjuvant may be administered systemically or locally. Moreover, the immuno-adjuvant may be administered before, after or simultaneous with the photosensitizing BPD. This permits the adjuvant-mediated activation/potentiation of immune responses to overlap with PDT mediated damage to tumor cells and any PDT induced immune responses.

After administration of the photosensitizer, sufficient time is permitted to elapse for the compound to be taken up by the tumor cells. This time for uptake may be varied according to various parameters, including but not limited to the photosensitizer administered, the route of administration, the physiology of the subject and of the tumor cells, and the artisan's skill and expeπence With green porphyπns, for example, the elapsed time may be from less than about one minute to more than about three hours, preferably from about one minute to about three hours, and more preferably from about 10 to about 60 minutes The cells, or tissue containing them, then are lπadiated at the wavelength of maximum absorbence of the photosensitizer In the case of BPDs, the wavelength is usually between about 550 and 695 nm, as discussed above In particular, red light is advantageous because of its relatively lower energy and the resulting lack of toxicity it poses to normal tissue while the tumor cells are destroyed

The compositions and methods of the present invention provide a useful immuno- adjuvant PDT treatment to treat, prevent or inhibit the development of metastatic tumors The following descπbes the compositions and formulations of the present invention and their clinical application Expeπmental data also are presented and descπbed

Since adjuvants may exert their activity by stimulating other agents that potentiate the development of an immune response, another aspect of the invention is the use of such agents in combination with PDT These agents include those that are immunomodulatory in activity and include several cytokines Examples of cytokines for use in the present invention are IL-12 and IL-18 (where "IL" refers to interleukin), granulocyte-macrophage colony stimulating factor (GM-CSF), and interferon-v (IFN-γ), which may be administered locally, systemically, or via expression vectors in combination with PDT

Another approach of the invention is to utilize a cytokine in combination with a factor that acts to promote the growth of hematopoietic progenitors in the presence of a cytokine FLT3 -ligand, isolated and cloned ia the corresponding FLT3 receptor [see refs Rosnet et -./ 1991, Matthews et al 1991, Rasko et al 1995, Lyman et al 1993, Lyman et al 1994] is an example of such a factor Alone, FLT3-hgand has relatively little activity but in combination acts synergistically with other cytokines including IL-3, IL-6, IL-7, IL- 1 1, IL-12 and colony stimulating factors to promote the growth of hematopoietic progenitors in vitro (Jacobsen et al 1995) Following the repeated administration of recombinant FLT3-hgand to mice, splenomegaly, hepatomegaly as well as substantial increases in spleen and blood myeloid progenitor activity were observed (Brasel et al 1996) indicating that FLT3-hgand mediates a mobilisation and expansion of hematopoietic stem cells.

Unexpectedly, mice given multiple FLT3-lιgand injections displayed dramatic increases in numbers of functionally mature dendπtic cells (DC) in multiple organs (Maraskovsky et al 1996, Shuπn et al 1997, Steptoe et al 1997) Bone maπow-deπved DC are potent APC that perform a sentinel role for the immune system These cells are normally present at low numbers within most tissues Their abundant expression of major histocompatibihty complex (MHC) gene products, adhesion and co-stimulatory molecules is a receptor repertoire that serves in the productive activation of naive and resting T lymphocytes (Steinman 1991 , Banchereau et al 1998) In association with T cells, DC may interact w ith and activate B cells and thereby regulate the formation of humoral immunity (Banchereau et al 1998) DC are significant sources of interleukin- 12 (IL-12), a pro-inflammatory cytokine that strongly promotes the formation of cellular immunity (Steinman 1991 , Banchereau et al 1998) In the generation of immune responses, DC are many times more effective than other APC types (B cells, macrophages) (Steinman 1991 , Banchereau et al 1998) Relatively few DC are required for the activation of large numbers of T cells In most tissues, DC are present in an undifferentiated state, inefficient at stimulating T cells However, these DC are highly efficient at captuπng antigen and the signals provided by antigen acquisition promotes a maturation process that yields DC that are highly effective at activating T lymphocytes DC phagocytose cells dying by apoptosis (programmed cell death), but not by necrosis (unregulated cell death), and can stimulate the expansion of numbers of antigen-specific cytotoxic T cells that recognize antigens contained withm apoptotic cells (Morse et al 1998, DiNicola et al 1998) In contrast, macrophages are incapable of processing apoptotic cells for the formation of specific cytotoxic T cell immunity (Morse et al 1998, DiNicola et al 1998) The capacity of DC to instigate de novo immune responses has lead to their designation as "nature 's adjuvant " (Steinman 1991 , Banchereau et al 1998, Young, et al 1996, Schuler et al 1997) Treatments that increase DC numbers and/or promote DC activation may ultimately foster specific T cell immunity Recent studies indicate that DC can provoke effective anti-tumour immunity m a vaπety of expeπmental systems In mice, effective immunity against solid tumours has been induced by pre-exposure of DC ex vivo to tumour-deπved peptides (Zitvogel et al 1996), crude cell extracts from non-immunogemc tumours (Flamand et al 1994), tumour cell-deπved mRNA (Ashley et al 1997, Boczkowski et al 1996), recombinant viral vectors (Song et al 1997, Specht et al 1997) or with DC-tumour cell fusions (Gong et al 1997) Further, it has been demonstrated that DC can stimulate cytotoxic T cell activity against leukemic cells and lymphoma (Choudhury et al 1997, Choudhury et al 1999, Fujii et -z/ 1999, Hsu et al 1996) DC exposed to tumour lysates or tumour-associated peptides in vitro had a vaccinating effect in human melanoma patients (Nestle et al 1998) The formation of specific cytotoxic (CD8+) T cell reactivity appears cπtical for effective anti-tumour immunity (Schuler et al 1997 , Morse et al 1998, DiNicola et al 1998) In cancer, vaπous factors may blunt the development of anti-tumour immunity This situation may aπse from

1 ) The action of soluble factors released by tumour cells that functionally impair immune cells.

2) Low or deficient expression of MHC or co-stimulatory molecules by tumour cells

3) A low capacity of tumour cells to present tumour-specific antigens to T cells

4) The loss of tumour-related antigens by tumour cell types

5) Tumour cell expression of receptors (e g Fas ligand) that compromise immune cell survival

DC are a unique immune cell population that is likely deπved from a myeloid linage precursor cell DC differentiation from bone marrow precursors is dπven by the cytokines GM-CSF and TNF-α (Bancheereau et al 1998) Additional cytokines including IL-4 and c-kit gand regulate the differentiation and maturation of DC at different developmental stages (Bancheereau et al 1998) After multiple FLT3-hgand injections, elevated DC numbers were found in immune and non-immune tissues including the spleen, peπpheral blood, thymus, liver, lungs, peπtoneal cavity, mesenteπc lymph nodes and Peyer's patches. These increases in DC numbers were approximately 17-fold in the spleen, 6-fold in the blood and 4-fold in peripheral lymph nodes. Importantly, these FLT3-ligand induced DC were as effective as splenic DC isolated from untreated mice in the induction of antigen-specific T cell responses. FLT3-ligand also modestly increased the number of natural killer (NK) cells in various regions (Shaw et al. 1998) and promoted the activation of NK in vivo by enhancing the interactions between DC and NK cells (Fernandez et al. 1999).

FLT3 -ligand treated mice implanted with syngeneic fibrosarcoma tumour cells, exhibited either no development of the tumour or a significantly lower tumour size (Lynch 1998). In vitro, FLT3-ligand had no direct effect upon tumour cell growth (Lynch 1998). FLT3-ligand produces a therapeutic effect against non-immunogenic tumours (Fernandez et al. 1999), murine melanoma (Esche et al. 1998), murine lymphoma (Esche et al. 1998) and limited the spread of metastases to the liver (Peron et al. 1998). The increased availability of DC in tumour-bearing FLT3-ligand-treated subjects may foster the recognition of tumour-associated structures by DC. The interaction of DC with NK cells may simulate NK cell-mediated tumour cell lysis releasing apoptotic or necrotic cell bodies that are taken up, transported, processed and presented by DC to T lymphocytes (Fernandez et al. 1999).

Thus the present invention includes the use of combined PDT/FLT3-ligand anti- cancer therapy. FLT3-ligand is currently available from Immunex (Seattle, Washington) as MOBIST™, while recombinant human and mouse FLT3-ligand is available commercially from the biological reagent supplier R&D (Minneapolis, Minnesota): Based on mouse studies, FLT3 -ligand may be adminstered to effect an increase in peripheral DC numbers. This may be accomplished by a regimen of regular administrations, such as a number of days for higher animals (e.g. humans). Standard PDT could be administered via intravenous injection of a photosensitiser followed later at a pre-determined time with light irradiation. FLT3-ligand administration may be continued for a number of days after PDT.

FLT3-ligand should be administered in a manner that when PDT is applied there is a high availability of DC within the body. When the delivery of PDT is co-ordinated with an FLT3-hgand-ιnduced zenith in DC numbers, the interaction of DC with dying tumour cells would be optimal This circumstance would provide the patient's immune system the greatest opportunity to generate a specific and effective response to tumour antigens - potentially providing the potential to limit residual and metastatic cancer through lmmunologic mechanisms

Yet another aspect of the invention involves a more direct use of dendπtic cell (DC) therapy m combination with PDT Since tumour cells may lack the capacity to directly stimulate T cell responses due to a lack of the appropπate repertoire of accessor} structures (MHC, co-stimulatory molecules, etc ) for instigating the responses, the acquisition of tumour cell material by DC could lead to the formation of specific anti- tumour immunity Thus the use of e\ vivo culture systems may circumvent immunosuppressive influences exerted by the tumour and permit the immune sensitisation to tumour antigens

One means of conducting this approach begins with a subject's peripheral blood DC being prepared and cultured in vitro for 24-48 hours with inactivated (optionally by PDT) tumor cells, tumor antigens, and/or any other tumor specific or related factor These DC, as antigen presenting cells, are re-introduced into the subject, with PDT applied to the subject either before or after the re-introduction

The Photosensitizers

The BPDs and green porphyπns useful in the method of the invention are descπbed in detail in Levy et al , U S Patent No 5,171,749 issued 15 December 1992, which is incorporated herein by reference "Green porphyπns" refer to porphynn denvatives obtained by reacting a porphynn nucleus with an alkyne in a Diels-Alder type reaction to obtain a monohydrobenzoporphynn Typically, green porphynns are selected from a group of porphynn deπvatives obtained by Diels-Alder reactions of acetylene denvatives with protoporphynn under conditions that promote reaction at only one of the two available conjugated, nonaromatic diene structures present in the protoporphyπn-IX nng system (πngs A and B) Several structures of typical green poφhyπns are shown in the above cited patent, which also provides details for the production of the compounds

Dimeπc forms of the green poφhyπn and dimeπc or multimenc forms of green porphyπn porphyπn combinations can be used The dimers and o gomenc compounds of the inv ention can be prepared using reactions analogous to those for dimeπzation and ohgomeπzation of poφhyπns er se The green poφhyπns or green poφhynn. poφhyπn linkages can be made directly, or poφhynns may be coupled, followed by a Diels-Alder reaction of either or both terminal poφhyπns to con ert them to the corresponding green poφhyπns

Additionally, the green poφhynn compounds used in the inv ention may be conjugated to v aπous gands to facilitate targeting to target tumor cells These hgands include those that are receptor-specific, or immunoglobulins as well as fragments thereof Preferred hgands include antibodies in general and monoclonal antibodies, as well as lmmunologically reactive fragments of both

The green poφhyπn compounds of the invention may be administered as a single compound, preferably BPD-MA, or as a mixture of various green poφhyπns Suitable formulations include those appropπate for administration of therapeutic compounds in vivo Additionally, other components may be mcoφorated into such formulations These include, for example, visible dyes or vaπous enzymes to facilitate the access of a photosensitizing compound to target tumor cells

Formulations

The photosensitizers and immuno-adju ants of the invention may be formulated into a variety of compositions These include posomes, nanoparticles, and pluromc (Poloxamer) containing formulations These compositions may also comprise further components, such as conventional delivery vehicles and excipients including isotonising agents, pH regulators, solvents, solubi zers, dyes, gelling agents and thickeners and buffers and combinations thereof Appropπate formulations and dosages for the administration of immuno-adjuvants are known in the art Suitable excipients for use with photosensitizers and immuno-adjuvants include water, saline, dextrose, glycerol and the like.

Typically, the photosensitizing agent is formulated by mixing it, at an appropriate temperature, e.g., at ambient temperatures, and at appropriate pHs, and the desired degree of purity, with one or more physiologically acceptable carriers, i.e., carriers that are nontoxic at the dosages and concentrations employed. Generally, the pH of the formulation depends mainly on the particular use, and concentration of photosensitizer, but preferably ranges anywhere from about 3 to about 8. Preferably, the photosensitizer is maintained at a pH in the physiological range (e.g., about 6.5 to about 7.5). The presence of salts is not necessary, and, therefore the formulation preferably is not an electrolyte solution. Appropriate nonantigenic ingredients, such as human serum albumin, may optionally be added in amounts that do not interfere with the photosensitizing agent being taken up by lens epithelial cells.

The particular concentration of a given BPD should be adjusted according to its photosensitizing potency. For example, BPD-DA can be used but at about a five-fold higher concentration than that of BPD-MA. Moreover, the BPD may be solubilized in a different manner than by formulation in liposomes. For example, stocks of BPD-MA or any other BPD may be diluted in DMSO (dimethylsulfoxide), polyethylene glycol or any other solvent acceptable for use in the treatment of tumors.

Normally, the adjustment of pH is not required when liposomal BPD-MA is used, as both components have a neutral pH. However, when other solvents than liposomes are used, the pH may require adjustment before mixing the BPD with the other material. Since antioxidants may interfere with the treatment, they should generally should be avoided.

Preparation of dry formulations that are reconstituted immediately before use also are contemplated. The preparation of dry or lyophilized formulations of the compositions of the present invention can also be effected in a known manner, conveniently from the solutions of the invention. The dry formulations of this invention are also storable. By conventional techniques, a solution can be evaporated to dryness under mild conditions, especially after the addition of solvents for azeotropic removal of water, typically a mixture of toluene and ethanol The residue is thereafter conveniently dπed, e g for some hours in a drying oven.

Suitable lsotomsmg agents are preferably nomonic isotonising agents such as urea, glycerol, sorbitol, manmtol, ammoethanol or propylene glycol as well as ionic isotonising agents such as sodium chloπde The solutions of this invention will contain the isotonising agent, if present, in an amount sufficient to bπng about the formation of an approximately isotomc solution The expression "an approximately isotomc solution" will be taken to mean in this context a solution that has an osmolanty of about 300 milhosmol (mOsm), conveniently 300 + 10 % mOsm It should be borne in mind that all components of the solution contnbute to the osmolanty The nomonic isotonising agent, if present, is added in customary amounts, I e . preferably in amounts of about 1 to about 3 5 percent bv weight, preferably in amounts of about 1 5 to 3 percent by weight

Solubihzers such as Cremophor types, preferably Cremophor RH 40, or Tween types or other customary solubihsers, may be added to the solutions of the invention in standard amounts

A further prefeπed embodiment of the invention relates to a solution compnsmg a BPD compound, and a partially etheπfied cyclodextnn. the ether substituents of which are hydroxyethyl, hydroxypropyl or dihydroxypropv 1 groups, a nomonic isotonising agent, a buffer and an optional solvent However, appropπate cvclodextnns should be of a size and conformation appropnate for use with the photosensitizing agents disclosed herein

Summaπes of pharmaceutical compositions suitable for use with the instant photosensitizers and immuno-adjuvants are known in the art and are found, for instance, in Remington's Pharmaceutical Sciences

Administration of Photosensitizers and Immuno- Adjuvants

As noted above, the treatment of the present invention is earned out m tissues either maligned with metatstatic tumors or susceptible to their occuπence, in an afflicted subject The photosensitizer and immuno-adjuvant containing preparations of the present invention may be administered systemically or locally and may be used alone or as components of mixtures Prefeπed routes of administration are intravenous, subcutaneous. intramuscular, or intraperitoneal injections of the photosensitizers and immuno-adjuvants in conventional or convenient forms. Injection of the adjuvant into a tumor, whether primary or resulting from metastasis, is preferred. Intravenous delivery of photosensitizers. is prefeπed, and intratumor injection may also be used when desired, as in pigmented tumor situations where the dose of PDT would be increased, for example. Oral administration of suitable oral formulations may also be appropriate in those instances where the photosensitizer may be readily administered to the tumor or tumor-prone tissue via this route.

The invention also includes the use of repeat treatments as deemed necessary by a suitable clinician or skilled worker in the field. Preferably, the treatment is repeated from 1 to about 10 times at intervals of about 1 to about 2 weeks. More preferably, the treatment is repeated from 1 to about 5 times, or most preferably for a total of 3 times, at approximately 2 week intervals.

Additionally, if the treatment is to be localized to an area of metastatic tumors suitable for topical formulations, the photosensitizers may be topically administered using standard topical compositions including lotions, suspensions or pastes.

The dose of photosensitizers and immuno-adjuvants can be optimized by the skilled artisan depending on factors such as, but not limited to, the physical delivery system in which it is carried, the individual subject, and the judgment of the skilled practitioner. It should be noted that the various parameters used for effective PDT in the invention are interrelated. Therefore, the dose should also be adjusted with respect to other parameters, for example, fluence, iπadiance, duration of the light used in PDT, and time interval between administration of the dose and the therapeutic iπadiation. One means of rapidly evaluating parameters for PDT/adjuvant administration is set forth below in Example 4. All of these parameters should be adjusted to produce significant damage to metastatic tumor cells and initiate an immune response without causing significant damage to the surrounding tissue. With photosensitizers, for example, the form of administration, such as in liposomes or when coupled to a target-specific ligand, such as an antibody or an immuno logically active fragment thereof, is one factor considered by a skilled artisan. Depending on the specificity of the preparation, smaller or larger doses of photosensitizers may be needed. For compositions which are highly specific to the target tumors, such as those with the photosensitizer conjugated to a highly specific monoclonal antibody preparation or specific receptor ligand, dosages in the range of 0 05-1 mg'kg are suggested For compositions which are less specific to the target, larger dosages, up to 1- 10 mg/kg, may be desirable The foregoing ranges are merely suggestive in that the number of vanables with regard to an individual treatment regime is large and considerable deviation from these values may be expected The skilled artisan is free to vary the foregoing concentrations so that the uptake and cellular destruction parameters are consistent with the therapeutic objectives disclosed above

The time of immuno-adjuvant deliver, may be before or after madiation with light as well as before or after administration of the photosensitizer, although madiation will occur after administration of the photosensitizer The immuno-adjuvant may be delivered immediately after madiation This may be of particular relevance with immuno-adjuvants that are opaque or otherwise interfere with irradiation

Without being bound by theory and in instances of BPDs being used as the photosensitizer, πτadiation is thought to result in the interaction of BPD in its triplet state with oxygen and other compounds to form reactive intermediates, such as singlet oxygen, which can cause disruption of cellular structures Possible cellular targets include the cell membrane, mitochondπa, lysosomal membranes

Each photosensitizer requires activation with an appropnate wavelength of light With BPDs, an appropπate light source, preferably a laser or laser diode, in the range of about 550 to about 695 nm, is used to destroy target cells An appropπate and prefeπed wavelength for such a laser would be 690-c 12 5 nm at half maximum. Generally, cell destruction occurs within 60 seconds, and likely is sufficiently complete within about 15 to about 30 seconds. The light dose administered dunng the PDT treatment contemplated herein can vary, but preferably ranges between about 10 to about 150 J/cm² The range between about 50-100 J/cm² is prefeπed. Increasing madiance may decrease the exposure times. Loca zed delivery of light is prefeπed, and delivery localized to the tumor is more preferred. Delivery of light pπor to photosensitizer activating light is also contemplated to improve penetration of the activating light. For example, irradiation of pigmented melanomas with infrared light before visible red light bleaches the melanin to improve penetration of the red light.

The time of light lπadiation after administration of the green poφhyπn may be important as one way of maximizing the selectivity of the treatment, thus minimizing damage to structures other than the target tumor cells Light treatment within about 3 hours before or after application of the photosensitizer should generally be attempted Alternatively, light treatment may be simultaneous, or nearly simultaneous, with said application

The following examples are intended to illustrate but not to limit the invention

Example 1 Sample Animals and Tumor Model Male, C57BL/6 mice were obtained from Charles River Canada (Montreal, QC) at 6 to 8 weeks of age. The B16-F0 and B16-F1 melanoma cell lines were obtained from the Amencan Type Tissue Collection (Manassas, Virginia) and grown as cell cultures in Dulbecco's Modified Eagle Medium (DMEM) (Gibco) supplemented with 10% fetal bovine serum (Sigma) The cells adhered to tissue culture plates, were removed for passage with 0.25% trypsin with 1.0 mM ethylenediaminetetraacetic acid (EDTA) (Gibco), and were cryo-preserved in liquid nitrogen in DMEM plus 40% FBS and 10% DMSO. Mice were injected with 5 X 10⁵ tumor cells in a total volume of 50 μL subcutaneously into the shaved, πght flank. The tumor size was monitored daily by measunng the diameter with vernier calipers and were treated when the tumors reached approximately 5 mm m diameter In initial experiments, the B16-F0 and B16-F1 were characterized with respect to in vivo growth rates and metastatic potential and were found to be identical. Subsequently the B16-F1 cell line was used for all expeπments. Example 2 Sample Immuno- Adjuvant PDT PDT treatment of mice beaπng the B 16-F1 tumor as performed as previously descπbed for the Ml rhabdomyosarcoma mouse tumor (Richter et al , 1987, Richter et al , 1988, Richter et al , 1991) Each mouse was weighed, warmed under infrared light for less than 5 min to dilate the blood vessels, restrained, and injected intravenously (tail vein) with Verteporfin at a concentration of 1 0 mg/kg bodv w eight using a 28G needle Thirty minutes later, animals were restrained and half of the animals were injected mtratumorally with 50 uL of Titermax adjuvant (Sigma) prepared as an emulsion w ith sterile phosphate buffered saline (PBS) according to the manufacturers specifications Animals ere then exposed to a light dose of 100 J/cm^: in a circular area encompassing the tumor of 1 cm diameter at 688 nm wavelength The power density as 70 mW/cπr and resulted in treatment times of 24 min per animal Following treatment, animals were monitored daily for tumor response

Example 3 Sample Expeπmental Metastases Pulmonary metastases were generated by intra enous injection of tumoi cells according to standard methods described by several groups (Chapoval et al , 1998. Lin et al , 1998, Volpert et al., 1998, Wang et al , 1998) Pulmonary metastases were initiated in each group of treated mice, as descnbed in Example 2 above, when the tumor was considered cured. This involved multiple treatments some of the mice and all test animals were injected intravenously with tumor cells on the same day Following PDT or immuno-adjuvant PDT animals were monitored for tumor response and if positive, Test (PDT and immuno-adjuvant PDT) and Control (naive) animals were injected with 5 X 10" tumor cells in 250 μl PBS via the lateral tail vein The animals were monitored for tumor recuπence and general health for 14 days after which the animals were sacπficed using CO, inhalation and their lungs removed Pulmonary metastases were clearly visible as black tumor colonies against the normal, pink lung tissue. Results from the above are shown in Figure 1. The B16 melanoma tumor model is inherently difficult to treat with PDT because of the absoφtion of light by the black melanin pigment secreted by the tumor cells. However, 10 animals completed the entire course of the experimental procedure. Five animals received PDT alone and of those animals, 3 required repeated PDT treatments to complete the tumor cure. Five animals received immuno-adjuvant PDT and 2 required second treatments with immuno-adjuvant PDT. All of the animals that had been treated with immuno-adjuvant PDT developed between 1 and 7 lung tumors at the time of dissection. One of the animals treated with PDT alone developed 6 lung colonies but the remaining 4 animals developed between 30 and 60 lung colonies. All of the control animals developed 200 to 300 lung colonies but the density of tumor growth made accurate quantification impossible (Fig. 1)

Thus immuno-adjuvant PDT evidently augments tumor immunity that develops duπng tumor growth and/or following PDT. Although the above example uses pigmented tumors in an expeπmental metastases approach, the results indicate that the combination of an immuno-adjuvant with PDT can be used for the treatment of metastatic cancer

Example 4 Rapid Evaluation of PPT/Adjuvant (PDV) Therapy via Lymphocyte Proliferation

In order to assess the potential usefulness of vaπous adjuvants and treatment parameters in PDV, an in vitro lymphocyte proliferation assay was designed and employed in a murine tumor model. The assay measures tumour-specific lymphocyte (tumor immunity) responses from animals treated with PDT and PDT combined with adjuvant (PDV). This permits the rapid evaluation of various PDT/adjuvant administration protocols.

Female C57B1/6 mice are implanted subcutaneously on the shaved right flank with the Lewis Lung Carcinoma (LLC) cell line. When tumours develop to approximately 5 mm diameter animals are treated with PDT or PDV. PDT is performed by delivering 1.0 mg/kg Verteporfm® i.v. 30 min prior to illumination of 125 J/cm2 delivered at 70 mW/cm2 (treatment time = 29 min, 4 sec). Animals treated with PDV receive a single 50 μl mtratumoral injection of adjuvant immediately following illumination Animals are monitored for general health and re-growth of the tumour following therapy

Seven to 10 days following therapy, animals are sacπficed and inguinal, axillary, cervical, and peπaortic lymph nodes are aseptically removed A single cell suspension is produced from the lymph nodes and this is cultured m half-area, 96-well tissue culture plates (Corning) in the presence of titrations of freeze/ hawed tumour cells and irradiated syngeneic splenocytes depleted of erythrocytes as accessory cells The cells are cultured in the presence of recombinant ιnterleukm-2 (Sigma), and concanavalin A (ConA) (Sigma) is utilized as a positive control to assess the prohferativ e capacity of lymphocytes Following 3 to 5 days of culture, the degree of proliferation is assessed using 3-(4,5- dιmethylthιazol-2-yl)-5-(3-carbo\ymethoxyphenyl)-2-(4-sulfophen l)-2H-tetrazohum, inner salt (Owen's reagent, MTS, from Promega), a variation of the MTT assav which produces a soluble formazan product which absorbs light at 490 nm The degree of proliferation is calculated by comparing the means of at least triplicate test wells to the means of lymphocytes cultured without antigen or mitogen (test mean - MTS background - control mean - MTS background x 100 = percent proliferation)

The assays may be performed using the commercial, expeπmental adju ant, Ribi Adjuvant System (RAS) (Coπxa) or Detox B-SE (Conxa) and alum for comparison

Of those animals treated with PDV which also responded to ConA (n=7), lymphocytes proliferated to 126 ± 19% (mean = standard deviation) of lymphocytes without antigen (see Fig 2) Animals treated with PDT alone proliferated to 108 3- 1 1% Controls using naive animals, tumour-beaπng animals treated with adjuvant alone, and proliferation in the presence of another syngeneic tumour to test specificity have also been tested

Example 5 Sample protocol for metastatic tumors

This protocol may be used for a vanety of metastatic tumors, including metastatic melanoma.

Liposomal verteporfin is injected at a dosage of 14 mg/m2 of body surface area, which is a higher dose than for treating AMD One to three hours later, diode laser light is applied at a rate of approximately 200mW/cm2 for a total dosage of 120-180J/cm2 to the lesion being treated. The dosage of the Detox adjuvant, which is injected into the lesion after PDT, provides in the range of 100-200μg of the cell wall skeleton component, and 20-30μg of the monophosphoryl lipid A component. This procedure is carried out at approximately 2 week intervals. Perferably there are 3 treatments.

All references cited hereinabove and below are hereby incoφorated by reference in their entireties, whether previously specifically incoφorated or not.

Having now fully described this invention, it will be appreciated by those skilled in the art that the same can be performed within a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation.

While this invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications. This application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth as follows in the scope of the appended claims.

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PARTICULATE ADJUVANTS

-exist as microscopic, insoluble particles

-generally, the immunogen must be incoφorated into or associated with the particle.

A. Mineral-based -insoluble, gel-like precipitate

-mineral formulations are the only adjuvants that are considered safe and effective for use in human vaccines i. Aluminum hydroxide (Alhydrogel)

Superfos chemicals http://www.superfos.com/index.htm a. SBAS4

Aluminum salt combined with monophosphoryl lipid A (MPL)

SmithKline Beecham http://www.sb.com/index.html ii. Aluminum phosphate (Adju-Phos) Superfos chemicals http://www.superfos.com/index.htm ii. Calcium phosphate

Superfos chemicals http://www.superfos.com/index.htm

B. Water-in-oil emulsions

-microdroplets of water, stabilized by surfactant in a continuous oil phase i. Freund's Complete Adjuvant (FCA) a mixture of a non-metabolizable oil (mineral oil), a surfactant (Arlacel A), and mycobacteria (M. tuberculosis or M. butyricum in Modified FCA) Superfos chemicals http://www.superfos.com/index.htm ii. Freund's Incomplete Adjuvant (FIA) has the same oil/surfactant mixture as FCA but does not contain any mycobacteria iii. Montanide Incomplete Seppic Adjuvant (ISA) Adjuvants a group of oil/surfactant based adjuvants in which different surfactants are combined with either a non-metabolizable mineral oil, a metabolizable oil, or a mixture of the two. They are prepared for use as an emulsion with aqueous Ag solution. The surfactant for Montanide ISA 50 is mannide oleate, a major component of the surfactant in Freund's adjuvants. The surfactants of the Montanide group undergo strict quality control to guard against contamination by any substances that could cause excessive inflammation, as has been found for some lots of Arlacel A used in Freund's adjuvant. The various Montanide ISA group of adjuvants are used as water- in-oil emulsions, oil-in-water emulsions, or water-in-oil-in-water emulsions. The different adjuvants accommodate different aqueous phase/oil phase ratios, because of the variety of surfactant and oil combinations. The performance of these adjuvants is said to be similar to Incomplete Freunds Adjuvant for antibody production; however the inflammatory response is usually less. Seppic, Paris, France

Oil-in-water emulsions

-microdroplets of squalene or squalane, stabilized with surfactants in a continuous water phase, developed for human clinical trials when combined with immunomodulators i. Ribi Adjuvant System (RAS)

4 components: (1) monophosphoryl lipid A (MPL); (2) trehalose dimycolate (TDM); (3) cell wall skeletons (CWS); (4) S. typhimurium mitogen (STM)

Ribi ImmunoChem Research, Inc.

ii. MF59 originally developed with N-acety-muramyl-L-alanyl-2-( ,2'- dipalmitolyl-sn-glycero-3'-phospho)ethylamide (MTP-PE) however when antibody titer was endpoint, MTP-PE was not required for adjuvant activity

Chiron Coφ. http://www.chiron.com/ iii. SBAS4 combination of monophosphory lipid A (MPL), QS21, and a proprietary oil in water emulsion

SmithKline Beecham http://www.sb.com/index.html iv. Detox™ active ingredients include MPL^® (derivative of the lipid A molecule found in gram negative bacteria) and mycobacterial cell wall skeleton

Corixa Coφoration http://www.corixa.com v. Detox B-SE™ for investigational use is supplied in clear glass vials.

Each vial contains: 145 micrograms CWS from M. phlei, 25 micrograms MPL from S. minnesota R595, 8.1 milligrams Squalane F, 0.38 milligrams Polysorbate 80 (USP/NF), 1.62 milligrams Soy Lecithin (NF), and 88 micrograms Sterile Water for Injection (USP)

Detox B-SE must be stored refrigerated between 2 and 8°C D. Immune stimulating complexes (ISCOM)

-open, cage-like structure resulting from the interaction of Quil-A with cholesterol and phosphatidycholine, human clinical trials

E. Liposomes

-single or multilamellar bilayer membrane vesicles comprised of cholesterol and phospholipid

-the immunogen may be membrane-bound or within the intermembrane spaces

F. Nano- and microparticles

-solid particles, biocompatible and biodegradable, synthetic polymers of cyanoacrylates, polycatide coglycohde (PLG) copolymer, antigen must be formulated with particle

NON-PARTICULATE ADJUVANTS

A. Muramyl dipeptide (MDP) and derivatives: Adjuvant peptides

-N-acetyl muramyl-L-alanyl-D-isoglutamine is the active component of peptidoglvcan extracted from Mycobacterium, derivatives are less toxic i. threonyl MDP ii. murabutide^V-acetylglucosaminyl-MDP (GMDP) a. Gerbu Adjuvant

Alternative to FCA. Oil is replaced by water-soluble, aliphatic quaternary amines or bio-degradable esterquats. Mycobacterium is replaced by GMDP. Gerbu Biotechnik GmbH, Gaiberg, Germany C-C Biotech 16766 Espola Road Poway, CA 92064 USA iii. murametide iv. nor-MDP

B. on-ionic block copolymers

-polymers composed of a region of hydrophobic polyoxypropylene (POP) flanked by regions of hydrophilic polyoxyethylene (POE), not biodegradable i. TiterMax

CytRx Coφoration http://www.cytrx.com/

iv. Syntex Adjuvant Formulation-1 (SAF-1)

Roche Bioscience (formerly Syntex Coφ., Palo Alto, CA) http://www.roche.com/pharma/Index.htm iv. SAF-2

C. Saponins

-extract of Quillaia saponaria tree, saponin is crude extract of triteφenoids i. Quil A

Partially purified saponin ii. Spikoside

Partially purified saponin iii. QS21 (Stimulon)

Purified, defined entity

Aquila Biopharmaceuticals, Inc. (formerly Cambridge Biotech Coφoration) http://www.aquilabio.com/ iv. ISCOPREP™ 703

Purified, defined entity

D. Lipid A and derivatives

-disaccharide of glucosamine with two phosphate groups and five or six fatty acid chains (C₁₂ to C₁₆ in length) i. monophosphoryl lipid A (MPL) removal of the 1 ' phosphate group from lipid A gives MPL

E. Cytokines

F. Carbohydrate polymers

-polymers of mannose and β l-3 glucose

-proposed as human vaccine adjuvants either mixed with or conjugated with immunogen

-stimulate macrophages and dendritic cells

G. Derivatized polysaccharides

-high molecular weight sulphated dextrans proposed as human vaccine adjuvants H. Bacterial toxins

-potent mucosal adjuvants in animal models

Claims

We claim:

1. A method of treating metastatic tumors in a subject, which method comprises: administering to a subject afflicted by metastatic tumors effective amounts of a photosensitizer and an immuno-adjuvant, and iπadiating said subject with light absorbed by said photosensitizer, wherein said method is photochemical mediated photodynamic therapy (PDT).

2. A method of preventing or inhibiting the development of metastatic tumors in a subject, which method comprises: administering to a subject at risk for developing metastatic tumors effective amounts of a photosensitizer and an immuno-adjuvant, and irradiating said subject with light absorbed by the photosensitizer.

3. A method of treating a primary tumor in a subject, which method comprises: administering to a subject clinically diagnosed with a primary tumor effective amounts of a photosensitizer and an immuno-adjuvant, and irradiating said subject with light absorbed by said photosensitizer.

4. The method of claim 2 wherein said subject has previously undergone cancer or tumor therapy.

5. The method of claims 1, 2 or 3 wherein said effective amount of a photosensitizer is in the range of 0.05 to 10 mg/kg.

6. The method of claim 5 wherein said effective amount of a photosensitizer is in the range of 0.05 to 1 mg/kg.

7. The method of claim 5 wherein said effective amount of a photosensitizer is in the range of 1 to 10 mg/kg.

8. The method of claims 1 or 3 wherein said photosensitizer is administered intravenously and said immuno-adjuvant is administered by injection into tumors.

9. The method of claims 1 or 3 wherein said iπadiation is localized to the tumors.

10. The method of claim 2 wherein said photosensitizer is administered intravenously or intratumorally.

11. The method of claims 1, 2 or 3 wherein said photosensitizer is administered, and the subject irradiated, before administration of the immuno-adjuvant.

12. The method of claims 1, 2 or 3 wherein said immuno-adjuvant is administered systemically.

13. The method of claims 1, 2 or 3 wherein the photosensitizer is a benzopoφhyrin derivative (BPD) or a green poφhyrin.

14. The method of claim 13 wherein the BPD is BPD-MA, EA6, or B3.

15. The method of claims 1, 2 or 3 further comprising an additional iπadiation, before iπadiation with light absorbed by the photosensitizer, with light of a wavelength which improves penetration of the absorbed light.

16. The method of claim 1 wherein said immuno-adjuvant comprises mycobacterial cell wall skeletons and de-3-O-acylated lipid A.

17. A pharmaceutical composition to treat, prevent, or inhibit the development of, metastatic tumors, said composition comprising: a photosensitizer and an immuno-adjuvant in amounts effective to treat, prevent, or inhibit the development of, metastatic tumors, and a pharmaceutically acceptable carrier or excipient.

18. The composition of claim 17 wherein the photosensitizer is a BPD or a green poφhyrin.

19. The composition of claim 18 which is a liposomal formulation.

20. The composition of claim 18 wherein the BPD is BPD-MA, EA6, or B3.

21. The composition of claim 18 wherein said immuno-adjuvant comprises mycobacterial cell wall skeletons and de-3-O-acylated lipid A.