Title:  Adjuvant incorporation into antigen carrying cells: compositions and methods

United States Patent:  6,218,166

Assignee:  John Wayne Cancer Institute (Santa Monica, CA)

Filed:  June 5, 1995

Disclosed are compositions and methods for enhancing the antibody and T cell response to cellular antigens by incorporating an immunopotentiating agent into the cellular membrane or into an intracellular compartment. Such adjuvant-incorporated cell compositions are useful in methods to increase immune responses against antigens, including immunologically cryptic tumor cell antigens, and may be employed to generate useful diagnostic antibodies, to elicit anti-tumor effects in immunized animals, and to significantly prolong survival in animals with cancer.

SUMMARY OF THE INVENTION

The present invention seeks to overcome these and other drawbacks inherent in the prior art by providing improved immunogenic compositions in which an adjuvant is incorporated into an intracellular compartment of, or incorporated onto or conjugated to, the outer membrane of an intact cell. Using such compositions, significantly improved immune responses are evoked. The invention may be employed to stimulate or increase the antibody or T cell responses against intracellular or membrane-bound antigens, even those that are otherwise poor immunogens. The invention also provides for the orchestration of cytokines in order to stimulate and activate cellular elements of the immune system.

Antibodies and T cells generated using the invention may be isolated from an animal and used in other methods, such as, particularly, using antibodies in a variety of diagnostic tests. Equally, the antibodies and T cells may continue to circulate in the animal in order to provide beneficial therapeutic effects.

In certain embodiments the invention concerns compositions, including pharmacologically acceptable formulations, that comprise one or more cells, or a population of cells, where the cells include one or more adjuvants associated with the cell surface or with an intracellular compartment. These compositions are referred to herein as adjuvant-conjugated cells or cellular complexes, adjuvant-incorporated cells or complexes, adjuvant-associated cells or complexes, or simply, "adjuvant-cell compositions".

Cells including one or more adjuvants "associated" with the cell surface, as used herein, means that the adjuvants are incorporated into or onto the membrane, conjugated to a membrane component, or otherwise physically and functionally associated with the intact membrane of a cell. The "physical and functional" association of the adjuvant with the membrane will be such that the adjuvant remains in contact with the membrane under physiological conditions in an amount effective to increase the immune response against cellular antigens present in the same membranous environment.

Cells including one or more adjuvants associated with "an intracellular compartment" means that the adjuvants are incorporated intracellularly, so that they are physically and functionally associated with an intracellular compartment of an intact cell. The adjuvants may be in contact with or located within any of the intracellular areas, including the cytoplasm, nucleus, golgi, endocytoplasmic reticulum, endocytotic vesicles and intracellular membrane surfaces, and also the mitochondria, ribosomes and other intracellular structures. The adjuvants will be present in amounts effective to increase the immune response against intracellular antigens of the cell, probably via processing and co-presentation of the adjuvant and intracellular antigen at the cell membrane.

The use of whole cells is an important feature of the invention that imparts many particular advantages. For example, tumor-associated antigens (TAAs) no longer have to be first identified or purified. omitting the purification step is a marked improvement in terms of time, difficulty and costs and, even more importantly, ensures that the antigens are presented in their natural environment. Isolation of TAAs has previously involved harsh conditions, such as extraction in 3 M KCl, which may destroy or modify certain of the epitopes. However, any extraction method that removes the TAA from the membrane environment is likely to alter its immunogenic properties, and it is an advantage of the present invention that this is no longer necessary.

The use of adjuvant-incorporated whole cells has the further advantages that the adjuvants are functionally associated with the antigens in the same membranous environment. This represents a more physiological-like situation over the general antigen-adjuvant admixtures used previously. As the immune system typically "sees" antigens presented at the surface of cells, incorporating the adjuvants into intact antigen-expressing cells more closely mimics host antigen presentation and will provide enhanced benefit to the animal.

The adjuvant-incorporated cell methodology of the invention has the further advantages that it is easier to generate immune responses against immunologically cryptic antigens. It is also simple, cost-effective, reproducible, and readily adaptable for use with any cell type. When administered to a human subject, it is also expected that the adjuvant-incorporated whole cells will prove effective at considerably lower doses with respect to the adjuvant itself. For example, in terms of MPL, it is contemplated that an effective response will result using MPL in the nanogram range, rather than the microgram range, such as the 10-500 .mu.gs proposed in U.S. Pat. No. 4,877,611.

As the present invention allows for the intracellular incorporation of adjuvants, it is also useful for generating or increasing immune responses against intracellular antigens. This is particularly advantageous as various tumor-associated antigens are intracellular. Numerous other antigens have intracellular locations, including various nuclear proteins, and even DNA, that are, for example, associated with certain autoimmune diseases.

The present invention provides a simple, but surprisingly effective method of preparing an adjuvant-incorporated cell complex, which method comprises incubating cells in an adjuvant-suspended culture media at an appropriate temperature and for a sufficient period of time, for example, as described herein in Example 5 and Example 8. The adjuvant-cell compositions of the invention, as exemplified by MPL-melanoma cell compositions, generally include between about 0.4 ng adjuvant, e.g., MPL, and about 3.1 ng adjuvant per 10⁶ cells; with levels of between about 1.6 ng adjuvant, e.g., MPL, and about 2.4 ng adjuvant per 10⁶ cells being routinely achieved.

The cell surface-associated adjuvants may be conjugated to any available membrane component, as exemplified by proteins, glycolipids and phospholipids in the membrane bilayer. Data is presented herein to show that the adjuvant-incorporated cells of the invention have adjuvants incorporated into the bilayer, and are not simply cells coated with, or loosely associated with, adjuvants. The adjuvant-incorporated cells of the invention are associated with effective amounts of adjuvants and yet the integrity of the cell is maintained.

Those working in the field of adjuvant compositions and their uses have previously experienced several difficulties due to the chemical properties of the adjuvants. For example, as certain adjuvants, such as MPL, are amphipathic they are difficult to solubilize in aqueous media. Furthermore, where adjuvants are intended for use in humans, which is usually the case, the use of many typical laboratory solvents, such as triethylamine or triethanolamine, and other liquids is not appropriate. The present inventors have discovered that such difficulties can be overcome by incubating the cells in an adjuvant-suspended culture media.

The "adjuvant-suspended culture media" of the invention are media, preferably, pharmacologically-acceptable media, that have been manipulated to contain higher amounts of one or more available adjuvants than normal. Particularly, the adjuvant-suspended culture media are capable of containing amphipathic adjuvants at levels higher than their normal solubility in aqueous media would suggest, and in a form so that they are "available" for conjugation to or association with the membrane of a cell, or for incorporation into an intracellular compartment. One advantageous manner of achieving this is to use one or more sonication cycles, as disclosed herein in Example 5.

The cellular components may be animal or human cells of virtually any type. Certain examples are cells infected with a virus that expresses a viral antigen at the cell surface and, also, tumor cells. Cells that secrete cytokines, or that are capable of secreting cytokines in response to certain stimuli, or cells that have been rendered capable of secreting cytokines due to genetic manipulation, i.e., altered by the hand of man, are also contemplated for use herewith. The use of cells that are capable of secreting cytokines and that also include one or more tumor-associated antigens in their membrane is also contemplated.

The use of tumor cells is preferred in certain cases. If desired, irradiated tumor cells may be employed to avoid significant adverse effects on the animal receiving the cells. Of course, irradiated tumor cells should always be employed in connection with human administration. The tumor cells may be irradiated prior to adjuvant conjugation, however, it is currently preferred that they be irradiated subsequent to the adjuvant conjugation process. Of course, irradiation could take place at any point of the preparative methods, or at several points.

A list of exemplary tumor cell lines is included herein, along with the corresponding ATCC designation numbers (Table 2 and Table 3). The cells in Table 3 will be preferred in certain embodiments. Tumor cells that have previously been used in animal studies or clinical trials will also be preferred, and any cell lines approved for human administration by the FDA will be particularly preferred.

Of course, other cells that have a particular antigen inserted into the membrane may also be used. This includes antigens physically mixed into or conjugated to the membrane, those incorporated following phagocytosis, and those expressed by the cell following insertion of a coding DNA segment into the cell ("recombinant cells"). In such ways, any antigen, such as a tumor antigen, may be expressed as the predominant antigen in a cell type that it is not normally associated with.

Cells that have been manipulated to incorporate other molecules in the membrane, such as additional targets or immunologically relevant molecules, may also be used. These include additional non-adjuvant protein components mixed into the membrane and molecules expressed by a cell following introduction of a coding DNA segment into the cell. Additional or different MHC class I or MHC class II antigens may thus be expressed in cells. It is particularly contemplated that a target molecule be included in the cell membrane to increase subsequent adjuvant-cell mediated or other host reactions against diseased cells of the animal. A currently preferred example is the addition of fibronectin in combination with an adjuvant to irradiated bladder cancer cells for use in bladder cancer treatment.

Tumor cells that express ganglioside antigens are a particular example of cells currently preferred for use in the present invention. Melanoma cells, including both mouse and human melanoma cells, are exemplary cells that have ganglioside antigens. The mouse melanoma cells termed B16, and the human melanoma cells termed M27, M18, M14, M111, M22, M7, M102, M108, M16, M104, M109, M14, M7, M25, M24, M10 and M101 are exemplary cell types for use in this invention; with M14, M7, M24, M25, M10 and M101 being preferred in certain cases; and M25, M10 and M101 currently being most preferred.

Tumor cells that include an antigen associated with an intracellular compartment are also contemplated for use in the present invention. Examples of important tumor-associated intracellular adjuvants are described in Example 8.

Although an understanding of the physical and chemical processes that operate during the conjugation method is not necessary to practice the invention, it is currently proposed that adjuvants such as MPL may preferentially bind to certain membrane proteins, such as proteins that are capable of binding to fatty acids. As such, it is contemplated that cells exhibiting higher levels of fatty acid binding proteins may be identified, or even developed, for advantageous use with the present invention.

Erythrocytes are a further example of cells also preferred for use in the present invention. It is contemplated that autologous erythrocytes would be obtained, e.g., from a cancer patient, and manipulated to form an adjuvant-incorporated cell, as disclosed herein. Erythrocytes obtained from cancer patients are known to be coated with tumor-associated antigens and can thus be used to promote an anti-tumor response. The use of erythrocytes has several advantages in that they are plentiful, easily obtained, do not require culturing and are a nuclear.

Compositions in accordance with the invention may include various cell types, one or more of which may include one or more adjuvants associated with the cell surface or an intracellular compartment, whilst other cells of the same type or other distinct cell types present may have no additional adjuvant. Particularly, multiple different adjuvants may be incorporated into a single cell or into multiple different cells. All such variations are within the scope of the invention. The use of multiple non-cell-associated adjuvants is well established in the art and such multiplicity is presently contemplated for use with the adjuvant-incorporated cells of the invention.

Virtually any adjuvant may also be employed in this invention, so long as the adjuvant may be incorporated into an intracellular compartment, or incorporated onto, physically associated with, or conjugated to the cell membrane of the cell in question, as described above. The adjuvants may be provided as purified components, in a partially purified state, or even as a membrane preparation or cellular extract, so long as the active components of such compositions can be incorporated into the cell itself or associated with, integrated into, or conjugated to the membrane of the target cell. Using membrane preparations and cellular extracts is not considered to be a particular problem due to the physical properties of the adjuvants and the mechanisms of membrane integration.

Adjuvants may also be chosen according to their documented properties. For example, adjuvants that are known to have both antibody- and T cell-enhancing capabilities will be preferred in certain cases. Adjuvants that stimulate the production of one, or preferably, of more than one, cytokine will also be preferred, particularly those that activate cytokines that stimulate further anti-tumor processes, such as by activating cytotoxic T cells (CTLs), NK cells, and the like. Adjuvants that particularly assist in inducing IgM antibodies are also preferred for use in certain embodiments.

A list of exemplary adjuvants for use in the present invention can be found herein in Table 1. Technic acids from Gram -ve cells, such as LTA, RTA, GTA, and their synthetic counterparts, are one preferred group of adjuvants. Hemocyanins and hemoerythrins, such as KLH, are also preferred, as are chitin and chitosan.

Another preferred group of adjuvants are the muramyl dipeptide (MDP) and tripeptide peptidoglycans and their derivatives, such as threonyl-NDP, fatty acid derivatives, such as MTPPE, and the derivatives described in U.S. Pat. No. 4,950,645, incorporated herein by reference. BCG, BCG-cell wall skeleton (CWS) and trehalose monomycolate and dimycolate (U.S. Pat. Nos. 4,579,945 and 4,520,019, each incorporated herein by reference) may also be used as adjuvants in the invention, either singly or in combinations of two or three agents, or in combination with MPL.

Amphipathic and surface active agents, such as QS21, and nonionic block copolymer surfactant form yet another group of preferred adjuvants. Quil A and lentinen are also preferred. Each of the above are generally non-toxic adjuvants or adjuvants with reduced toxicity and are preferred for human administration.

A particularly preferred adjuvants are currently the lipopolysaccharide (endotoxin) group of adjuvants. This is exemplified by lipid A, which may be used in animals, and detoxified endotoxins, which may be used in animals and humans. Detoxified and refined endotoxins, and combinations thereof, are described in U.S. Pat. Nos. 4,866,034; 4,435,386; 4,505,899; 4,436,727; 4,436,728; 4,505,900.

Further preferred adjuvants are the bacterial superantigens. Although useful in all aspects of the invention, these adjuvants are particularly contemplated for use in generating or enhancing the immune response against intracellular antigens, including intracellular tumor antigens. Examples of bacterial superantigens include Staphylococcal enterotoxins, e.g., as produced by S. aureus and S. epidermidis; E. coli exotoxins, and other membrane proteins and toxins from bacteria such as Clostridium perfringens and Streptococcus pyogenes.

The non-toxic derivative monophosphoryl lipid A (MPL) is currently the most preferred adjuvant, as it has both antibody-and T cell-enhancing capabilities and because it induces IgM antibodies. MPL derivatives and synthetic MPLs, as described by Johnson et al. (1990), Grabarek et al. (1990), Baker et al. (1992; 1994); Tanamoto et al. (1994a;b; 1995); Brade et al. (1993) and U.S. Pat. No. 4,987,237, each incorporated herein by reference, form a particularly preferred group of adjuvants for incorporation into cells.

Adjuvants that do have certain toxic side effects may still be used in this invention, however, so long as their toxicity is monitored and kept within the limits generally known to acceptable as side effects. In experimental animals, these limits will be higher than in humans. Indeed, toxicity and other adverse effects are less problematic where animals are used, e.g., for the purposes of raising antibodies or T cells specific for a cellular antigen.

To prepare an adjuvant-incorporated cell composition in accordance with the present invention, one will generally first prepare an adjuvant-suspended composition, preferably by sonication, and then admix a composition of the cells with the adjuvant suspension. The admixture is then incubated at a suitable temperature and for a period of time to effect intracellularly incorporation or membrane conjugation. Exemplary suitable conditions are described herein in Example 5 and Example 8.

Although a wide variety of other conjugation methods are possible, using a sonication cycle to suspend an amphipathic adjuvant in an aqueous medium is most preferred as this is believed to result in amounts of adjuvant conjugated per cell that could not previously be achieved. Increasing the conjugation temperature above 4^oC. is also preferred as this results in an increase of intracellular transport of the adjuvant, as shown in Example 8. Temperatures between about 10^oC. and about 40^oC. are preferred, with the physiological temperature of about 37^oC. being most preferred for use with human cells.

The amount of adjuvant conjugated to a cell may generally be increased or maximized in order to optimize the immunopotential of the composition. As disclosed herein in Example 5 and Example 8, by way of example only, incubating cells with a sonicated MPL suspension at a level of about 75 .mu.g of MPL per million cells results in levels of between about 0.4 ng and about 3.1 ng of cell surface-associated MPL per million cells.

The methods associated with the invention include methods for generating an antibody or a T cell response, which methods generally comprise administering to an animal an immunologically effective amount of an adjuvant-incorporated cell composition that includes at least one cell type that includes at least one adjuvant associated with the cell surface or with an intracellular compartment. Immunologically effective amounts are those amounts effective to stimulate cytokine production, or preferably, to stimulate an antibody or antigen-specific T cell response in the animal, as exemplified herein.

A sample or samples may subsequently be obtained from the animal to provide the desired antibodies or T cells. Blood samples are appropriate for obtaining polyclonal antibodies and less-purified T cells compositions, whereas spleen cells may be obtained to provide monoclonal antibodies and more purified T cell compositions. The methods for generating purified antibodies and T cells from such samples are well known to those of skill in the art, and are further described in the present disclosure.

The compositions administered to animals or humans in these methods may include additional components in combination with the adjuvant-incorporated cell compositions, such as immunologically effective amounts of purified antibodies, activated lymphocytes and/or other cells of a different or the same type. The cells may be obtained from the animal or human in question, e.g., allogenic tumor cells or erythrocytes. The lymphocytes may also be obtained from the animal or human in question and activated in vitro using known techniques.

The methods of the invention may be used, in certain circumstances, as therapy for diseases, such as cancer, where the stimulation of the immune system enhances the anti-tumor activity of the animal. As such, the invention also provides methods for stimulating therapeutic or otherwise beneficial immune responses in an animal with a disease, such as methods for stimulating an anti-tumor cell immunological response in an animal having any one of a variety of cancers. The MPL-incorporated cells of the invention may also be used to inhibit the onset of septicemia and endotoxemia, as described in U.S. Pat. No. 4,844,894.

These methods comprise, generally, administering to such an animal a pharmacologically-acceptable adjuvant-incorporated cell composition comprising cells that include a tumor antigen and that are manipulated to have one or more adjuvants associated with the cell surface or with an intracellular compartment. The cells are administered in an amount effective to stimulate an antibody or T cell response against the diseased cells of the animal. The tumor antigen-containing adjuvant-incorporated cell compositions may include irradiated tumor cells or erythrocytes, preferably those obtained from the animal to be treated. Currently, about 24 million adjuvant-incorporated melanoma cells are contemplated for use in melanoma therapy, with each cell containing at least about 1 ng of adjuvant or more. Any pharmacologically-acceptable medium may be used, as are widely available commercially.

Where tumor cells are used, they may be autologous tumor cells, i.e., obtained from the same animal or human patient that is to be treated and then inactivated outside the body. Alternatively, the tumor cells may be allogenic cells, in which case they will generally include an intracellular or membrane-bound tumor antigen that stimulates an antibody or T cell response against a tumor antigen of the tumor cells of the animal to be treated, i.e., they will be generally of the same tumor type and will stimulate a "cross-reactive" immune response.

The tumor antigen-containing adjuvant-incorporated cell compositions may be administered to an animal or patient by any acceptable method, including injection into the general circulation or injection into the tumor site. The administration of such cells, whether of human or animal origin, having adjuvants in their outer membranes may be used to achieve effective cancer therapy in human patients, as disclosed herein. Treatment methods of the invention may be used after, prior to, or in combination with other methods, such as, radiotherapy and chemotherapy. Pre-treatment with BCG (Bast et al., 1974; Bennet et al., 1988; Minden et al., 1976; Yamamoto et al., 1988) is contemplated to be particularly useful.

Claim 1 of 103 Claims

What is claimed is:

1. A composition comprising a cell that includes an adjuvant non-covalently incorporated into the cell surface membrane or an intracellular compartment of said cell.

____________________________________________
If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.