The invention provides compositions and methods for treating or preventing a target disease in a mammal by induction of memory T cells. The methods comprise (1) administering a sensitizing composition comprising a non-target antigen to a patient; and (2) administering a therapeutic composition comprising a non-target antigen and a target antigen to the patient, wherein the target antigen is associated with the disease, and wherein the therapeutic composition is administered after the sensitizing compositions, at an interval sufficient for induction of memory T cells. The methods can alternatively comprise administering a target antigen along with an inhibitory agent such as an inhibitor of DC apoptosis, an inhibitor of Granzyme B, a Granzyme-B-inducible mediator of apoptosis, an inhibitor of perforin, or a perforin-inducible mediator of apoptosis.

Description of the Invention

BACKGROUND OF THE INVENTION

Vaccines have long been used for the prevention of infectious diseases, such as viral or microbial infections. The cell-mediated arm of the immune system is extensively involved in providing the host with the ability to defend, recover from infections and to prevent further infections by the same antigen.

Cell-mediated immune mechanisms are also thought to be useful against other diseases such as cancer and autoimmune diseases. Several tumor-related antigens have been identified and used to prepare vaccines intended to treat cancer (e.g., lineage-specific antigens such as MART-1, tyrosinase, gp100, cancer-testis antigens, such as Her2/Neu, CEA, or antigens overexpressed on rapidly proliferating cells, such as cyclins, survivin, etc.). In addition to the defined antigens and their epitopes, whole tumor cells are also used as the source of cancer-relevant antigens.

There is a need for more efficient therapeutic vaccines and improved methods of treatment of cancer and autoimmune diseases.

BRIEF SUMMARY OF THE INVENTION

The invention provides methods of treating or preventing a target disease in a mammal by induction of activated T cells. Use of a non-target antigen to sensitize a patient's CD8.sup.+ T cells can be followed with administration of a vaccine comprising a target antigen (such as a tumor-associated antigen or a pathogen-associated antigen) as well as the non-target antigen.

In one aspect, the invention provides a method of treating or preventing a target disease comprising (1) administering a sensitizing composition comprising a non-target antigen to a patient; and (2) administering a therapeutic composition comprising a non-target antigen and a target antigen to the patient, wherein the target antigen is associated with the disease, and wherein the therapeutic composition is administered after the sensitizing compositions, at an interval sufficient for induction of memory T cells.

In another aspect, the invention provides a method of treating or preventing a target disease comprising (1) determining the presence of memory-type immunity to one or more non-target antigens in a patient; and (2) administering a therapeutic composition comprising the non-target antigen and a target antigen to the patient, wherein the target antigen is associated with the disease and wherein the patient was found to have memory-type immunity to the non-target antigen in step (1).

In a further aspect, the invention provides a method of treating or preventing a target disease comprising (1) determining immunity to one or more non-target antigens in a patient; (2) isolating a sample of dendritic cells from the patient; (3) loading the dendritic cells with a target antigen and a non-target antigen, wherein the patient was found to have immunity to the non-target antigen in step (1); and (4) administering to the patient a therapeutic composition comprising the loaded dendritic cells.

In yet another aspect, the invention provides a composition for treating a disease comprising a target antigen, one or more non-target antigens, and a pharmaceutically acceptable carrier.

In another aspect, the invention provides a kit for the treatment of a disease comprising multiple therapeutic compositions, each therapeutic composition comprising a target antigen and one or more non-target antigens.

In yet another aspect, the invention provides a method of treating or preventing a target disease comprising administering a therapeutic composition comprising a target antigen and an inhibitory agent, wherein the target antigen is associated with the target disease, and wherein the inhibitory agent is an agent for preventing the killing of dendritic cell or other antigen-presenting cells, such as an inhibitor of DC apoptosis, an inhibitor of Granzyme B, a Granzyme-B-inducible mediator of apoptosis, an inhibitor of perforin, or a perforin-inducible mediator of apoptosis.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides methods and compositions for the treatment or prevention of a disease in a patient by the induction of memory T cells, using a combination of a target antigen (such as a tumor-associated antigen or a pathogen-associated antigen) and a non-target antigen. Without being bound by any particular theory, it is thought that CD8.sup.+ memory T cells to a non-target antigen can support dendritic cells (DC) in activating additional naive CD8.sup.+ T cells against a target antigen, thereby increasing cytotoxic T lymphocyte (CTL) activity against cells having the target antigen, such as tumor cells or cells infected with a pathogen.

In some embodiments, the invention provides a method of treating or preventing a target disease comprising (1) administering a sensitizing composition comprising a non-target antigen to a patient; and (2) administering a therapeutic composition comprising a non-target antigen and a target antigen to the patient, wherein the target antigen is associated with the disease, and wherein the therapeutic composition is administered after the sensitizing compositions, at an interval sufficient for induction of memory T cells.

The interval required for induction of memory T cells can be evaluated using standard methods known to one of ordinary skill in the art and described herein. For example, the levels of CD62L or CCR7 can be detected using flow cytometry assays (Sallusto et al., Annu Rev Immunol 22: 745-763 (2004); Lanzavecchia et al., Science 290:92-97 (2000). Observation of CD62L(high) or CD45R0(high)/CD62L(high) phenotype on the expanded population of antigen specific CD8.sup.+ T cells compared to the effector stage where CD62L (negative/low cells predominate can indicate that memory T cell activity has been induced. Additionally or alternatively, high expression of a surface marker CCR7 (see, e.g., Rivino et al., J. Exp. Med. 200:725-735 (2004)) or low levels of intracellular marker Granzyme B in the expanded antigen-specific population can be used to identify the memory cells with functional helper function (see, e.g., FIG. 4 (see Original Patent) and Nakamura et al., Cancer Res 67:10012-10018 (2007) at FIG. 1 (see Original Patent)). Although naive cells express similar to memory cells CD62L (high), CCR7 (high), and Grenzyme B (low)phenotype, in contrast to memory cells they are not expanded (low frequencies) and do not express CD45R0 marker, instead expressing CD45RA (Lanzavecchia et al., Nat Rev Immunol 2: 982-987 (2002)). In other embodiments, one of ordinary skill in the art may simply employ a pre-determined interval between administration of the sensitizing composition and a therapeutic composition. For example, an interval of 4 weeks, 5 weeks, 6 weeks, or more than 6 weeks may separate administration of compositions comprising a particular non-target antigen. It will be understood that in some patients and/or in the use of different non-target antigens, a shorter or longer interval may be advantageous.

The sensitizing composition can comprise one or more non-target antigens. Preferably, the non-target antigen of the sensitizing composition is the same as the non-target antigen of the therapeutic composition. In some embodiments, the sensitizing composition comprises multiple non-target antigens, and the therapeutic composition comprises at least one of the non-target antigens present in the sensitizing composition. In other embodiments, the sensitizing composition comprises multiple non-target antigens, and the therapeutic composition also comprises multiple non-target antigens. The sensitizing composition and/or the therapeutic composition can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more than 20 non-target antigens.

The non-target antigen can be any suitable epitope against which CD8.sup.+ T cells can be safely and desirably be induced. The non-target antigen can be viral, bacterial, protozoan, helminthic, mycobacterial, alloantigen, or xenoantigen. In a preferred embodiment, the epitope can be from a viral source, such as hepatitis Herpes simplex, CMV, EBV and other herpes viruses, HBV and HCV, and influenza virus. Any suitable non-target antigen or combination thereof can be used, although it will be understood that commercially available compositions, such as influenza vaccines, will be particularly useful in the methods of the present invention.

The target disease can be any disease such as cancer or an infectious disease. In one preferred embodiment, the disease is cancer. The cancer can be of any organ or tissue, including but not limited to solid organ tumors. For example, the cancer can be melanoma, colon-, breast-, lung, cervical-, ovarian, endometrial-, prostate-, skin-, brain-, liver-, kidney, thyroid, pancreatic, esophageal-, or gastric cancer, leukemias, lymphomas, multiple myeloma, myelodysplastic syndrome, premalignant HPV-related lesions, intestinal polyps and other chronic states associated with increased cancer risk. It will be understood that known or later discovered cancer-associated antigens can be used in the compositions and methods of the present invention as target antigens for treating cancer.

In another embodiment, the target disease is an infectious disease. The infectious disease can have a viral, bacterial, protozoan, parasitic, or other pathogenic origin. In some preferred embodiments, the disease can be primarily caused by or secondary to infections such as HIV, hepatitis B, hepatitis C, cytomegalovirus (CMV), Epstein-Barr virus (EBV), Pox virus, influenza including avian flu, severe acute respiratory syndrome (SARS), mycobacterial infections including tuberculosis and leprosy, leishmaniasis, malaria, anthrax and other bioterrorism agents. It will be understood that known or later discovered pathogen-associated antigens can be used in the compositions and methods of the present invention as target antigens for treating infectious diseases.

The patient can be a human or any suitable non-human mammal such as a mouse, rat, rabbit, cat, dog, pig, sheep, cow, or primate. In some embodiments, the patient is a non-human experimental animal model. In a preferred embodiment, the patient a primate. In a more preferred embodiment, the patient is a human.

In some embodiments, the method can further include the step of administering one or more additional therapeutic compositions each comprising the target antigen and a non-target antigen. In such embodiments, each non-target antigen or combination thereof can be distinct, i.e., although each therapeutic composition comprises a common target antigen and one or more non-target antigens, no therapeutic composition comprises the same non-target antigen(s) present in any other therapeutic composition. In a preferred embodiment, multiple therapeutic compositions are administered cyclically, i.e., no single therapeutic composition is administered consecutively.

The invention further provides a method of treating or preventing a target disease comprising (1) determining the presence of memory-type immunity to one or more non-target antigens in a patient; and (2) administering a therapeutic composition comprising the non-target antigen and a target antigen to the patient, wherein the target antigen is associated with the disease and wherein the patient was found to have memory-type immunity to the non-target antigen in step (1).

The presence of memory-type immunity to one or more non-target antigens can be determined, as described above, by any method known to one of ordinary skill in the art, such as by determining CD62L levels or DC killing capacity.

In some embodiments, the method can further comprise the step of administering to the patient one or more additional therapeutic compositions as described above, comprising the target antigen and a non-target antigen, wherein the patient was determined to have immunity to the each non-target antigen in step (1).

Where memory-type immunity to a non-target antigen is determined prior to administration of a therapeutic compound, a sensitizing composition is not necessarily employed, but if a sensitizing composition is to be administered, it can include any suitable non-target antigen or combination thereof as described above.

Additionally, the invention provides a method of treating or preventing a target disease comprising (1) determining immunity to one or more non-target antigens in a patient; (2) isolating a sample of dendritic cells from the patient; (3) loading the dendritic cells with a target antigen and a non-target antigen, wherein the patient was found to have immunity to the non-target antigen in step (1); and (4) administering to the patient a therapeutic composition comprising the loaded dendritic cells.

In some embodiments, the method can further comprise the step of administering to the patient one or more additional therapeutic compositions comprising dendritic cells loaded with the target antigen and a non-target antigen, wherein the patient was determined to have immunity to each non-target antigen in step (1).

The invention further provides a composition for treating a target disease comprising a target antigen, one or more non-target antigens, and a pharmaceutically acceptable carrier.

The target disease, target antigen and/or non-target antigen can be as described above. In some embodiments, one or more of the antigens are covalently linked. Preferably, a target antigen is linked to a non-target antigen.

In yet another embodiment, the invention provides a method of treating or preventing a target disease comprising administering a therapeutic composition comprising a target antigen and an inhibitory agent. The inhibitory agent can be any agent useful for converting suppressor activity of effector cells into helper cells. Without being bound by any particular theory, it is thought that such a combination can prevent the killing of dendritic cells or other antigen-presenting cells. In some embodiments, the inhibitory agent(s) can be inhibitors of DC apoptosis, inhibitors of Granzyme B, Granzyme-B-inducible mediators of apoptosis, inhibitors of perforin, and perforin-inducible mediators of apoptosis. (see, e.g., Maczek et al., Int J Cancer 115: 450-455 (2005); Semenzato et al., Cancer 48: 2191-2197 (1981); Tsukishiro et al., Cancer Immunol Immunother 52: 599-607 (2003)).

In some embodiments, the inhibitor is covalently linked to the antigen. The inhibitory agent and antigenic determinant can be linked directly, by a linker sequence, or any form of a carrier molecule. In a preferred embodiment, the inhibitor and the target antigen are linked together and linked to additional molecules facilitating their entry into cells. The inhibitory agent can optionally be linked to the target antigen and/or to additional molecule(s), such as molecules capable of facilitating entry into cells.

The compositions of the present invention can be prepared in any useful pharmaceutical formulation known to one of ordinary skill in the art. It will be understood that in many embodiments, the compositions will be in liquid form. The liquid compositions of the invention, whether they be solutions, suspensions or other like form, can include one or more of the following components: sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono or diglycerides which can serve as the solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. Physiological saline is a preferred diluent. An injectable pharmaceutical composition is preferably sterile.

The pharmaceutical compositions can be prepared by methodology well known in the pharmaceutical art. The antigens of the invention can be in the form of a solvate in a pharmaceutically acceptable solvent such as water or physiological saline. Alternatively, the compounds can be in the form of the free base or in the form of a pharmaceutically acceptable salt such as the hydrochloride, sulfate, phosphate, citrate, fumarate, methanesulfonate, acetate, tartrate, maleate, lactate, mandelate, salicylate, succinate and other salts known in the art. The appropriate salt would be chosen to enhance bioavailability or stability of the compound for the appropriate mode of employment (e.g., oral or parenteral routes of administration).

Dendritic cells used as live vaccines can be injected in saline, phosphate buffer, or other cell media supporting their viability. In some embodiments, the media can be supplemented with human plasma, human serum, or human serum albumin.

A composition formulated to be administered by injection can be prepared by combining the agonist with water, and preferably buffering agents, so as to form a solution. The water is preferably sterile pyrogen-free water. A surfactant can be added to facilitate the formation of a homogeneous solution or suspension. Surfactants are compounds that facilitate dissolution or homogeneous suspension of the agonist in the aqueous delivery system. Other carriers for injection include, without limitation, sterile peroxide-free ethyl oleate, dehydrated alcohols, propylene glycol, as well as mixtures thereof.

Suitable pharmaceutical excipients for the injecting solutions include stabilizing agents, solubilizing agents, buffers, and viscosity regulators. Examples of these adjuvants include ethanol, ethylenediaminetetraacetic acid (EDTA), tartrate buffers, citrate buffers, and high molecular weight polyethylene oxide viscosity regulators. These pharmaceutical formulations can be injected intramuscularly, epidurally, intraperitoneally, or intravenously.

The invention also provides a kit for the treatment of a target disease comprising multiple therapeutic compositions as described above, each therapeutic composition comprising a target antigen and one or more non-target antigens.
 

Claim 1 of 21 Claims

1. A method of vaccinating a patient against a target antigen comprising: (a) administering a sensitizing composition comprising a non-target antigen to a patient; and (b) administering a therapeutic composition comprising a non-target antigen and the target antigen to the patient, wherein the non-target antigen in the sensitizing composition is the same as or different than the non-target antigen in the therapeutic composition, and wherein the therapeutic composition is administered after the sensitizing composition, at an interval sufficient for induction of memory T cells, whereby the patient is vaccinated against the target antigen.
 

____________________________________________
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.