HK1179518A - Method of treatment using ligand-immunogen conjugates - Google Patents

Description

Methods of treatment with ligand-immunogen conjugates

This application is a divisional application of an invention patent application entitled "method of treatment with ligand-immunogen conjugates" filed 3/30/2001 under the national application number 01810185.2(PCT/US 01/10254).

Technical Field

The present invention relates to methods and pharmaceutical compositions for treating diseases characterized by the presence of pathogenic cell populations. More specifically, the cell-targeting ligand-immunogen complex is administered to the diseased host, preferably in combination with an immune system stimulant or other therapeutic factor, to enhance and/or redirect the host's immune response to the pathogenic cells.

Background

The mammalian immune system provides a tool for the recognition and elimination of tumor cells, other pathogenic cells, and invading foreign pathogens. Although the immune system normally provides a strong line of defense, there are many instances where cancer cells, other pathogenic cells or pathogens evade the host's immune response and proliferate or persist with and accompany host morbidity. Chemotherapeutic agents and radiation therapy have been developed to eliminate replicating tumors. However, most, if not all, of the currently available chemotherapeutic agents and radiation treatment regimens have toxic side effects because they act to not only destroy cancer cells, but also affect normal host cells, such as cells of the hematopoietic system. In addition, chemotherapeutic agents have limited efficacy when the host develops resistance.

Foreign pathogens may also proliferate in a host by evading an effective immune response or in the event that the host immune system is not responding due to medication or other health issues. Although a number of therapeutic compounds have been developed, a number of pathogens are, or have become, resistant to such therapeutic agents. The ability of cancer cells and infectious organisms to develop resistance to therapeutic agents, and the toxic side effects of currently available anticancer agents, make it highly desirable to develop new therapies that are specific for pathogenic cell populations and have reduced host toxicity.

Researchers have developed therapeutic protocols for destroying cancer cells by specifically targeting cytotoxic compounds to such cells. These protocols utilize toxins conjugated to ligands that bind to receptors that are either specific to cancer cells or overexpressed, in an attempt to minimize the transmission of the toxin to normal cells. Using this approach, immunotoxins consisting of antibodies directed against specific receptors on pathogenic cells, in association with toxins such as ricin, pseudomonas exotoxin, diphtheria toxin and tumor necrosis factor, have been developed. These immunotoxins target tumor cells bearing specific receptors recognized by the antibody (Olsnes, S., immunol. today, 10, p. 291-295, 1989; Melby, E.L., Cancer Res., 53(8), p. 1755-1760, 1993; Better, M.D., 1991, PCT publication WO 91/07418, published 5/30).

Another approach to selectively target a population of cancer cells or an exogenous pathogen in a host is to enhance the host's immune response against the pathogenic cells, thereby eliminating the need to administer compounds that may also exhibit independent host toxicity. One reported strategy for immunotherapy is to bind antibodies, such as genetically engineered poly-antibodies, to the surface of tumor cells to display their constant regions on the cell surface to induce tumor cell killing through various immune system-mediated processes (De Vita, v.t., biological Therapy of Cancer, 2 nd edition, philiadelphia, Lippincott, 1995; Soulillou, j.p., U.S. patent 5,672,486). However, this approach is complicated by the difficulty in determining tumor-specific antigens. Another approach that is dependent on the immunological activity of the host is to target anti-T cell receptor antibodies or anti-Fc receptor antibodies to the surface of tumor cells to promote direct binding of immune cells to the tumor (Kranz, d.m., U.S. patent 5,547,668). A vaccine-based approach is also described which relies on a vaccine comprising an antigen fused to a cytokine which modifies the immunogenicity of the vaccine antigen, thereby stimulating an immune response against the pathogen (pilai, s., PCT publication WO 91/11146 published 2/7 1991). The method relies on indirect modulation of the reported immune response. Another method for killing unwanted cell populations utilizes the Fab fragment of IL-2 or anti-thymocyte globulin in combination with an antigen to eliminate unwanted T cells; however, based on reported experimental data, this approach appears to eliminate only 50% of the target cell population and results in non-specific cell killing in vivo (i.e., 50% of peripheral blood lymphocytes that are not T cells are also killed (poulty, p., published PCT publication WO97/37690 on 16/10/1997)). Thus, there remains a significant need for therapies directed at treating disease states characterized by the presence of pathogenic cell populations in the affected host.

The present invention relates to a method of eliminating pathogenic cell populations in a host by enhancing the host immune system in recognizing and responding to such cell populations. Effectively enhancing the antigenicity of said cellular pathogen to enhance the endogenous immune response mediated elimination of the population of pathogenic cells. The method avoids or reduces the use of cytotoxic or antimicrobial therapeutic agents. The method comprises administering a ligand-immunogen conjugate wherein the ligand is capable of specifically binding to a population of pathogenic cells that uniquely express, preferentially express, or overexpress a ligand binding moiety in vivo, and the ligand-conjugated immunogen is capable of eliciting antibody production, or more preferably is capable of being recognized by endogenous or co-administered exogenous antibodies in the host animal. Immune system-mediated elimination of pathogenic cells is directed by binding of the immunogen-conjugated ligand to a receptor, transporter, or other surface-presenting protein uniquely expressed, overexpressed, or preferentially expressed by the pathogenic cells. The surface-presented protein uniquely expressed, overexpressed, or preferentially expressed by the pathogenic cells is a receptor that is not present or is present in lower amounts on non-pathogenic cells, providing a method for selectively eliminating the pathogenic cells. At least one additional therapeutic factor, such as an immune system stimulant, a cell killing agent, a tumor penetration enhancer, a chemotherapeutic agent, a cytotoxic immune cell, or an antimicrobial agent, may be co-administered to the host animal to enhance the therapeutic efficacy.

In one embodiment, the method of the invention comprises the steps of: administering a ligand capable of specifically binding with high affinity in vivo to a cell surface protein uniquely expressed, preferentially expressed or overexpressed on said target population of pathogenic cells, said ligand being conjugated to an immunogen against which an innate or adaptive immunity already present or which may be provoked in said host animal is directed, optionally co-administering at least one therapeutic factor which is an endogenous immune response activator or cytotoxic compound. In a preferred embodiment, the method comprises administering to the host animal a ligand-immunogen conjugate composition, wherein the ligand is folate or another folate receptor binding ligand. The ligand is conjugated, e.g., by covalent binding, to an immunogen capable of eliciting an antibody response in the host animal, or more preferably to an immunogen that binds to an existing endogenous antibody (resulting from innate or acquired immunity) or a co-administered antibody (i.e., by passive immunization) in the host animal. At least one additional therapeutic factor, cell killing agent, tumor penetration enhancer (e.g., inflammatory or pro-inflammatory factor), chemotherapeutic agent, cytotoxic immune cell, or antimicrobial agent that does not specifically bind to the ligand-immunogen complex but is capable of stimulating or enhancing the endogenous immune response can be administered to the host animal in conjunction with the administration of the ligand-immunogen conjugate.

In accordance with another embodiment of the present invention, there is provided a method of enhancing the endogenous immune response-mediated specific elimination of a population of pathogenic cells in a host animal harboring said population, wherein members of said population have binding sites accessible to a ligand. The method comprises the following steps: administering to the host a ligand-immunogen conjugate composition comprising a complex of the ligand and an immunogen known to be recognized by endogenous or exogenous antibodies in the host or known to be directly recognized by immune cells of the host, and at least one additional composition comprising a therapeutic factor selected from the group consisting of a cell killing agent, a tumor penetration enhancer, a chemotherapeutic agent, an antimicrobial agent, a cytotoxic immune cell, and a compound capable of stimulating an endogenous immune response, wherein the compound does not bind to the ligand-immunogen conjugate.

In accordance with an alternative embodiment of the present invention, there is provided a method of enhancing an endogenous immune response-mediated specific elimination of a population of pathogenic cells in a host animal harboring said population, wherein said population expresses a binding site for a ligand. The method comprises the following steps: administering to said host a composition comprising a complex of said ligand and an immunogen; administering to said host antibodies directed against said immunogen; and administering to the host at least one additional therapeutic factor selected from the group consisting of a cell killing agent, a tumor penetration enhancer, a chemotherapeutic agent, an antimicrobial agent, a cytotoxic immune cell, and an endogenous immune response stimulator that does not bind the ligand-immunogen complex.

In a preferred embodiment of the invention, there is provided a method of enhancing the endogenous immune response-mediated specific elimination of a population of pathogenic cells in a host animal harboring said population, wherein said population preferentially expresses, uniquely expresses or overexpresses a folate receptor. The method comprises the following steps: administering to the host a composition comprising a covalently linked conjugate of an immunogen and a ligand, wherein the immunogen is known to be recognized by endogenous or exogenous antibodies in the host, or the immunogen is known to be recognized directly by immune cells of the host, and the ligand comprises folic acid or a folic acid analog having a glutamyl group, wherein the covalent linkage to the immunogen is through only the gamma-carboxyl group of the glutamyl group. In another embodiment, at least one additional composition comprising a therapeutic factor selected from the group consisting of a cell killing agent, a tumor penetration enhancer, a chemotherapeutic agent, an antimicrobial agent, a cytotoxic immune cell, and a compound capable of stimulating an endogenous immune response is administered to the host, wherein the compound does not bind to the ligand-immunogen conjugate.

In yet another embodiment of the present invention, a method is provided for enhancing the endogenous immune response-mediated specific elimination of a population of pathogenic cells in a host animal harboring said population, wherein said population preferentially expresses, uniquely expresses, or overexpresses a folate receptor. The method comprises the following steps: administering to the host a composition comprising a covalently linked conjugate of an immunogen and a ligand, wherein the immunogen is known to be recognized by endogenous or exogenous antibodies in the host, or the immunogen is known to be recognized directly by immune cells of the host, and the ligand comprises folic acid or a folic acid analog having a glutamyl group, wherein the covalent linkage to the immunogen is through only the alpha-carboxyl group of the glutamyl group. In another embodiment, at least one additional composition comprising a therapeutic factor selected from the group consisting of a cell killing agent, a tumor penetration enhancer, a chemotherapeutic agent, an antimicrobial agent, a cytotoxic immune cell, and a compound capable of stimulating an endogenous immune response is administered to the host, wherein the compound does not bind to the ligand-immunogen conjugate.

In yet another embodiment of the invention, the target population of pathogenic cells is a population of cancer cells. In another embodiment, the target cell population is virally infected endogenous cells. In another embodiment, the target cell population is a population of an exogenous organism, such as a bacterium, mycoplasma, yeast, or fungus. The ligand-immunogen conjugates bind to the surface of tumor cells or pathogenic organisms and "label" the cell membrane of the target cell population with the immunogen, thereby triggering an immune-mediated response against the labeled cell population. Antibodies administered to the host in passive immunization or antibodies present in the host system as a result of existing innate or acquired immunity bind to the immunogen and trigger an endogenous immune response. The antibody binds to the cell-bound ligand-immunogen conjugate, causing complement-mediated cytotoxicity, antibody-dependent cell-mediated cytotoxicity, antibody opsonization and phagocytosis, antibody-induced receptor aggregation that signals cell death or rest, or any other humoral or cellular immune response stimulated by the binding of the antibody to the cell-bound ligand-immunogen conjugate. In cases where immune cells can directly recognize antigens without prior antibody opsonization, direct pathogenic cell killing can occur.

Elimination of exogenous pathogens or infected endogenous cells or tumor endogenous cells can be further enhanced by administration of therapeutic factors, cell killers, tumor penetration enhancers, chemotherapeutic agents, cytotoxic immune cells, or antimicrobial agents capable of stimulating an endogenous immune response. In one embodiment, the cytotoxic immune cell is a population of cytotoxic immune cells that are isolated, expanded ex vivo, and then injected into a host animal. In another embodiment of the invention, an immunostimulant is used, said immunostimulant being an interleukin such as IL-2, IL-12 or IL-15 or an IFN such as IFN- α, IFN- β or IFN- γ or GM-CSF. In another embodiment, the immunostimulatory substance may be a cytokine composition comprising a combination of cytokines, e.g., IL-2, IL-12, or IL-15 in combination with IFN- α, IFN- β, or IFN- γ, or GM-CSF, or any effective combination thereof, or any other effective combination of cytokines.

In yet another embodiment of the present invention, a pharmaceutical composition is provided comprising a therapeutically effective amount of a ligand-immunogen conjugate, a therapeutic factor selected from the group consisting of a cell killing agent, a tumor penetration enhancer, a chemotherapeutic agent, an antimicrobial agent, a cytotoxic immune cell, and a compound capable of stimulating an endogenous immune response, and a pharmaceutically acceptable carrier therefor, the ligand-immunogen conjugate being capable of specifically binding to a population of pathogenic cells in a host animal and promoting specific elimination of said cells either by an acquired or innate immune response, a co-administered antibody, or directly by immune cells in said host, wherein said compound is not bound to said ligand-immunogen conjugate. In one embodiment, the pharmaceutical composition is in a parenteral sustained release dosage form. In another embodiment, the therapeutic factor is an immunostimulatory substance comprising a compound selected from the group consisting of: interleukins such as IL-2, IL-12, IL-15, IFNs such as IFN-alpha, IFN-beta or IFN-gamma and GM-CSF or combinations thereof.

Detailed Description

Methods of therapeutically treating a host having cancer or a host infected with a pathogenic organism are provided. The methods result in enhancing immune response-mediated elimination of the population of pathogenic cells by conferring/tagging antigenicity to the pathogenic cells, causing the cells to be recognized and eliminated by the host immune system. The methods utilize ligand-immunogen conjugates that are capable of high affinity binding to cancer cells or other pathogens. The high affinity binding may be intrinsic to the ligand, which may be improved (enhanced) with chemically modified ligands or due to specific chemical bonding between the ligand and the immunogen present in the conjugate. The methods can also utilize combination therapies employing the ligand-immunogen conjugates and additional therapeutic factors, cell killing agents, chemotherapeutic agents, tumor penetration enhancers, cytotoxic immune cells, or antimicrobial agents capable of stimulating an endogenous immune response to enhance immune response-mediated elimination of the population of pathogenic cells.

The methods of the invention can be used to enhance endogenous immune response-mediated elimination of a population of pathogenic cells in a host animal harboring the population of pathogenic cells. The present invention is applicable to populations of pathogenic cells that cause a wide variety of conditions, such as cancer and infectious diseases. Thus, the pathogenic cell population may be a tumorigenic cancer cell population, including benign tumors and malignant tumors, or it may be non-tumorigenic. The cancer cell population may be generated spontaneously, obtained by mutation such as present in the germ line of the host animal or somatic mutation, or it may be induced chemically, virally, or radioactively. The invention may be used to treat cancers such as carcinomas, sarcomas, lymphomas, hodgkin's disease, melanomas, mesotheliomas, burkitt's lymphoma, snuff cancer, leukemias, and myelomas. The cancer cell population may include, but is not limited to, oral cancer, thyroid cancer, endocrine cancer, skin cancer, gastric cancer, esophageal cancer, laryngeal cancer, pancreatic cancer, colon cancer, bladder cancer, bone cancer, ovarian cancer, cervical cancer, uterine cancer, breast cancer, testicular cancer, prostate cancer, rectal cancer, kidney cancer, liver cancer, and lung cancer.

The pathogenic cell population can also be a foreign pathogen or a cell population carrying a foreign pathogen, such as a virus. The invention is applicable to foreign pathogens such as bacteria, fungi, viruses, mycoplasma and parasites. The pathogens that can be treated with the present invention can be any infectious organism known in the art to cause disease in an animal, including organisms such as: gram-negative or gram-positive coccal or bacillal bacteria, DNA viruses, and RNA viruses, including, but not limited to, DNA viruses such as papillomaviruses, parvoviruses, adenoviruses, herpesviruses, and vaccinia viruses, and RNA viruses such as arenaviruses, coronaviruses, rhinoviruses, respiratory syncytial viruses, influenza viruses, picornaviruses, paramyxoviruses, reoviruses, retroviruses, and rhabdoviruses. Of particular interest are antibiotic-resistant bacteria, such as antibiotic-resistant streptococci (Streptococcus species) and staphylococci (staphylococcus species), or bacteria that are sensitive to antibiotics but cause recurrent infections with antibiotic treatment, such that resistant organisms eventually develop. Such organisms can be treated with the ligand-immunogen conjugates of the present invention in combination with antibiotics at doses lower than those normally administered to patients to avoid the development of these antibiotic-resistant bacterial strains. The invention is also applicable to any fungus, mycoplasma species, parasite or other infectious organism that is pathogenic in an animal. Examples of fungi that can be treated by the method of the invention include fungi that grow as molds or yeasts, including, for example, fungi that cause diseases such as: tinea, histoplasmosis, blastomycosis, aspergillosis, cryptococcosis, sporotrichosis, coccidioidomycosis, paracoccidioidomycosis, and candidiasis. The present invention may be used to treat parasitic infections, including but not limited to infections caused by: somatic tapeworm, schistosome, histiocytosis, amoeba and Plasmodium (Plasmodium), Trypanosoma (Trypanosoma), Leishmania (Leishmania) and Toxoplasma species. Parasites of particular interest are those that express the folate receptor and bind folate; however, there is a large body of reference in the literature for ligands that exhibit high affinity for infectious organisms. For example, penicillins and cephalosporins known for their antibiotic activity and specific binding to bacterial cell wall precursors can likewise be used as ligands for the preparation of ligand-immunogen conjugates for use in accordance with the present invention. The ligand-immunogen conjugates of the present invention may also be directed against a population of cells bearing an endogenous pathogen, wherein the pathogen-specific antigen is preferentially expressed on the surface of cells bearing the pathogen, and serves as a receptor for a ligand that specifically binds to the antigen.

The methods of the invention may be used in human clinical medicine and veterinary applications. Thus, the host animal harboring the population of pathogenic organisms and treated with the ligand-immunogen conjugates can be a human, or in the case of veterinary applications, a laboratory animal, an agricultural animal, a domesticated animal, or a wild animal. The present invention may be applied to host animals including, but not limited to: a human being; laboratory animals such as rodents (e.g., mice, rats, hamsters, etc.), rabbits, monkeys, chimpanzees; domestic animals such as dogs, cats, and rabbits; farm animals, such as cattle, horses, pigs, sheep, goats; and wild animals kept in captivity, such as bears, pandas, lions, tigers, leopards, elephants, zebras, giraffes, gorillas, dolphins and whales.

The ligand-immunogen conjugate is preferably administered parenterally to the host animal, for example, intradermally, subcutaneously, intramuscularly, intraperitoneally, or intravenously. Alternatively, the conjugate can be administered to the host animal by other pharmaceutically useful methods, and any effective dose and suitable therapeutic dosage form, including sustained release dosage forms, can be used. The methods of the invention may be used in combination with surgical removal of tumors, radiation therapy, chemotherapy, or biological therapy such as other immunotherapies including, but not limited to: monoclonal antibody therapy, treatment with immunomodulatory drugs, adoptive transfer of immune effector cells, treatment with hematopoietic growth factors, cytokines, and vaccination.

According to the present invention, the ligand-immunogen conjugates may be selected from a wide variety of ligands and immunogens. The ligand must be capable of specifically eliminating the population of pathogenic cells in the host animal because the pathogenic cells preferentially express receptors for the ligand that are accessible for binding. Acceptable ligands include folic acid, folic acid analogs and other folate receptor binding molecules, other vitamins, peptide ligands identified by library screening, tumor-specific peptides, tumor-specific aptamers (aptamers), tumor-specific carbohydrates, tumor-specific monoclonal or polyclonal antibodies, Fab or scFv (i.e., single chain variable region) fragments of antibodies such as Fab fragments of antibodies directed against EphA2 or other proteins specifically expressed or specifically accessible on metastatic cancer cells, small organic molecules from combinatorial libraries, growth factors such as EGF, FGF, insulin and insulin-like growth factors and homologous polypeptides, somatostatin and analogs thereof, transferrin, lipoprotein complexes, bile salts, selectins, steroid hormones, peptides containing Arg-Gly-Asp, retinoids, various galectins (galectins), delta-opioid receptor ligands, Cholecystokinin a receptor ligands, angiotensin AT1 or AT2 receptor specific ligands, peroxisome proliferator activated receptor gamma ligands, beta-lactam antibiotics, small organic molecules including antimicrobials, and other molecules that specifically bind to receptors preferentially expressed on the surface of tumor cells or infectious organisms, or fragments of any of these molecules. In the case of ligands that bind to infectious organisms, of interest are any molecules known in the art to preferentially bind to the microorganism, such as antibiotics or other drugs. The invention is also applicable as a ligand (e.g., an antimicrobial agent), or other cell surface protein, for a molecule designed to fit the binding pocket of a particular receptor, based on the crystal structure of the receptor, and wherein the receptor is preferentially expressed on the surface of tumors, bacteria, viruses, mycoplasma, fungi, parasites, or other pathogens. In a preferred embodiment of the invention, it is also contemplated that binding of the ligand to any tumor antigen or other molecule preferentially expressed on the surface of tumor cells may be utilized.

The binding site for the ligand may include receptors for any molecule capable of specifically binding to a receptor, wherein the receptor or other protein is preferentially expressed on a population of pathogenic cells, including receptors for, for example, growth factors, vitamins, peptides (including opioid peptides), hormones, antibodies, carbohydrates, and small organic molecules. The binding site may also be that of any molecule, such as an antibiotic or other drug, where it is known in the art that such sites are preferentially present on microorganisms. For example, the binding site may be that of a beta-lactam antibiotic (e.g., penicillin) in the bacterial cell wall, or that of an antiviral agent present specifically on the surface of the virus. The present invention is also applicable to binding sites for ligands (e.g., antimicrobial drugs) designed to fit the binding site of the receptor based on the crystal structure of the receptor, and wherein the receptor is preferentially expressed on the surface of the pathogenic cell or organism. It is also envisaged that tumour specific antigens may be used as binding sites for ligands in the methods of the invention. One example of a tumor specific antigen that can be used as a binding site for ligand-immunogen conjugates is an extracellular epitope of a member of the Ephrin family of proteins (e.g., EphA 2). EphA2 expression was restricted to normal cell-cell junctions, but in metastatic tumor cells EphA2 was distributed over the entire cell surface. Thus, EphA2 on metastatic cells would be accessible for binding to, for example, an immunogen-conjugated antibody Fab fragment, while the protein would be inaccessible for binding to Fab fragments on normal cells, resulting in a ligand-immunogen conjugate specific for metastatic cancer cells. The present invention also contemplates the use of ligand-immunogen conjugates in combination to maximize targeting of the pathogenic cells for elimination by either an acquired or innate immune response or by co-administered antibodies.

Acceptable immunogens for use in the present invention are immunogens that are capable of eliciting antibody production in a host animal, or that have previously elicited antibody production in a host animal, caused existing immunity, or are part of the innate immune system. Alternatively, antibodies to the immunogen can be administered to the host animal to establish passive immunity. Suitable immunogens for use in the present invention include antigens or antigenic peptides against which existing immunity has been raised by normal scheduled vaccination or prior natural exposure to factors such as poliovirus, tetanus, typhus, rubella, measles, mumps, pertussis, tuberculosis and influenza antigens and alpha-galactosyl groups. In such a case, the ligand-immunogen conjugate will serve to redirect the previously obtained humoral or cellular immunity to a population of pathogenic cells within the host animal to eliminate the foreign cells or pathogenic organisms. Other suitable immunogens include antigens or antigenic peptides against which the host animal has been raised by a new immunity generated by immunization against an unnatural antigen or hapten (e.g., fluorescein isothiocyanate or dinitrophenyl), as well as antigens against which an innate immunity exists (e.g., superantigens and muramyl dipeptide).

The ligands and immunogens of the present invention may be conjugated using any method known in the art for forming complexes. This may include covalently, ionically or hydrogen bonding the ligand to the immunogen either directly or through a linking group (e.g., a divalent linker). The conjugate is typically covalently linked through the ligand to the immunogen through the formation of an amide, ester or imino bond (imino bond) between an acid, aldehyde, hydroxyl, amino or hydrazo group on the corresponding component of the complex. In a preferred embodiment of the invention, the ligand is folic acid, an analog of folic acid, or any other folate receptor binding molecule, and a folic acid ligand conjugated to the immunogen by a process for preparing gamma-esters of folic acid via a pteroyl azide intermediate using trifluoroacetic anhydride. This preferred method results in the synthesis of a folate ligand conjugated to the immunogen only via the gamma-carboxyl group of the glutamic acid group of folate, wherein the gamma-conjugate binds with high affinity to the folate receptor, avoiding the formation of a mixture of alpha-and gamma-conjugates. Alternatively, pure α -conjugates can be prepared from intermediates in which the γ -carboxyl group is selectively blocked to form α -conjugates, followed by deblocking of the γ -carboxyl group using organic synthesis protocols and methods well known in the art. In particular, other vitamins may be used as ligands for preparing the conjugates according to the invention. For example, biotin and riboflavin and folic acid can be used to form ligand-immunogen conjugates (see U.S. Pat. nos. 5,108,921, 5,416,016 and 5,635,382, incorporated herein by reference).

The ligand-immunogen conjugates of the present invention enhance the endogenous immune response-mediated elimination of pathogenic cell populations. The endogenous immune response may include a humoral response, a cell-mediated immune response, and any other immune response endogenous to the host animal, including complement-mediated cell lysis, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody opsonization leading to phagocytosis, production of apoptotic signals upon antibody binding, anti-proliferative or differentiated receptor aggregation, and direct immune cell recognition of the delivered antigen/hapten. It is also contemplated that the endogenous immune response will be secreted using cytokines that regulate processes such as immune cell proliferation and migration. The endogenous immune response may include the involvement of immune cell types such as B cells, T cells (including helper and cytotoxic T cells), macrophages, natural killer cells, neutrophils, LAK cells, and the like.

The humoral response may be a response induced by processes such as normally scheduled vaccination or active immunization with a natural antigen or a non-natural antigen or hapten (e.g., fluorescein isothiocyanate) that induces a new immunity. Active immunization involves multiple injections of non-native antigens or haptens outside of the normal immunization schedule to induce new immunity. The humoral response may also result from innate immunity in which the host animal has a natural existing immunity, e.g., immunity to alpha-galactosyl groups. Alternatively, passive immunization can be established by administering to the host animal an antibody, such as a natural antibody collected from serum or a monoclonal antibody which may or may not be a genetically engineered antibody (including humanized antibodies). The use of a specific amount of antibody reagent to generate passive immunity, and the use of ligand-immunogen conjugates in which passively administered antibodies are directed against the immunogen, will provide the advantage of a standard set of reagents used in cases where the patient's existing antibody titers against other potential antigens are not therapeutically useful. Passively administered antibodies can be "co-administered" with the ligand-immunogen conjugate, co-administration being defined as administration of the antibody prior to, concurrently with, or subsequent to the administration of the ligand-immunogen conjugate.

It is contemplated that the existing antibody, induced antibody, or passively administered antibody will be redirected to the invasive cells or organisms by preferentially binding the ligand-immunogen conjugate to the tumor cells or infectious organisms, thereby killing the pathogenic cells via complement-mediated lysis, ADCC, antibody-dependent phagocytosis, or antibody aggregation of receptors. The cytotoxic process may also involve other types of immune responses such as cell-mediated immunity, and secondary responses that are produced when attracted antigen-presenting cells phagocytose the unwanted cells and deliver natural tumor antigens or foreign pathogen antigens to the immune system to eliminate the cells or organisms bearing the antigens.

At least one additional composition comprising a therapeutic factor may be administered to the host in combination with, or as an adjuvant to, the methods detailed above to enhance the endogenous immune response-mediated elimination of the population of pathogenic cells, or more than one additional therapeutic factor may be administered. The therapeutic factor may be selected from a compound capable of stimulating an endogenous immune response, a chemotherapeutic agent, an antimicrobial agent, or other therapeutic factor capable of compensating for the efficacy of the administered ligand-immunogen complex. The process of the invention can be carried out by: in addition to administering the above conjugates, a compound or composition capable of stimulating an endogenous immune response including, but not limited to, the following factors is administered to the host: cytokines or immunocytogrowth factors, such as interleukins 1-18, stem cell growth factor, basic FGF, EGF, G-CSF, GM-CSF, FLK-2 ligand, HILDA, MIP-1 α, TGF β, M-CSF, IFN α, IFN β, IFN γ, soluble CD23, LIF, and combinations thereof.

Therapeutically effective combinations of these cytokines may also be used. In a preferred embodiment, for example, a therapeutically effective amount, e.g., IL-2 in an amount ranging from about 5000 IU/dose/day to about 500,000 IU/dose/day (in a daily multiple dose regimen) and IFN- α in an effective amount, e.g., in an amount ranging from about 7500 IU/dose/day to about 150,000 IU/dose/day (administered in a daily multiple dose regimen), are used with folate-linked fluorescein isothiocyanate to eliminate such cell populations in host animals harboring pathogenic cell populations. In another preferred embodiment, a therapeutically effective amount of IL-12 and IFN- α is used, and in yet another preferred embodiment, a therapeutically effective amount of IL-15 and IFN- α is used. In an alternative preferred embodiment, IL-2, IFN- α or IFN- γ and GM-CSF are used in combination. Preferably, the therapeutic factors used, such as IL-2, IL-12, IL-15, IFN- α, IFN- γ and GM-CSF (including combinations thereof) activate natural killer cells and/or T cells. Alternatively, the therapeutic factor or combination thereof (including interleukins in combination with interferon and GM-CSF) may activate other immune effector cells, such as macrophages, B cells, neutrophils, LAK cells, and the like. The present invention also contemplates the use of any other effective combination of cytokines, including other combinations of interleukins and interferons and colony stimulating factors.

Chemotherapeutic agents that are cytotoxic in nature and can be used to enhance tumor permeability suitable for use in the methods of the present invention include adrenocorticoids, alkylating agents, antiandrogens, antiestrogens, androgens, estrogens, antimetabolites such as cytarabine, purine analogs, pyrimidine analogs, and methotrexate, busulfan, carboplatin, chlorambucil, cisplatin and other platinum compounds, tamoxifen, paclitaxel, cyclophosphamide, plant alkaloids, prednisone, hydroxyurea, teniposide, antibiotics such as mitomycin C and bleomycin, nitrogen mustards, nitrosureas, vincristine, vinblastine, inflammatory and pro-inflammatory agents, and any other chemotherapeutic agent well known in the art. Other therapeutic agents that may be adjunctive to administration of the conjugates of the invention include penicillin, cephalosporin, vancomycin, erythromycin, clindamycin, rifampin, chloramphenicol, aminoglycoside, gentamicin, amphotericin B, acyclovir, trifluridine, ganciclovir, zidovudine, amantadine, ribavirin and any other antimicrobial compound known in the art.

Elimination of the pathogenic cell population will include reduction or elimination of the tumor mass or pathogenic organism, resulting in a therapeutic response. In the case of a tumor, the elimination may be the elimination of primary tumor cells or cells that have metastasized or are in the process of detaching from the primary tumor. In accordance with the present invention, prophylactic treatment to prevent recurrence of tumors after removal by any treatment method, including surgical removal of the tumor, radiation therapy, chemotherapy or biological therapy, is also contemplated. The prophylactic treatment can be a primary treatment with the ligand-immunogen conjugate (e.g., in a daily multi-dose regimen) and/or can be an additional treatment or series of treatments spaced days or months after the primary treatment.

The invention also relates to pharmaceutical compositions comprising an effective amount of a ligand-immunogen conjugate to "label" a population of pathogenic cells in a host animal for specific elimination by endogenous immune response or by co-administered antibodies. The composition further comprises an additional agent selected from the group consisting of a cell killing agent, a tumor penetration enhancer, a chemotherapeutic agent, an antimicrobial agent, a cytotoxic immune cell, and a compound capable of stimulating an endogenous immune response, in an amount effective to enhance elimination of the pathogenic cells, wherein the compound is not bound to the ligand-immunogen conjugate. The pharmaceutical composition contains a therapeutically effective amount of the ligand-immunogen conjugate and the therapeutic factor, which may include cytokines such as IL-2, IL-12 or IL-15 or a combination of cytokines including IL-2, IL-12 or IL-15 and interferons such as IFN- α or IFN- γ, as well as a combination of interferons, interleukins and colony stimulating factors such as GM-CSF.

The unit daily dosage of the ligand-immunogen conjugate may vary widely depending upon the physical condition of the host, the condition to be treated, the molecular weight of the conjugate, its route of administration and tissue distribution, and the possibility of co-using other therapeutic treatments, such as radiation therapy. The effective amount administered to the patient is based on the body surface area, the weight of the patient, and the physician's assessment of the condition of the patient. An effective amount may range from about 1ng/kg to about 1mg/kg, more preferably from about 1 μ g/kg to about 500 μ g/kg, and most preferably from about 1 μ g/kg to about 100 μ g/kg.

Any effective regimen of administering the ligand-immunogen conjugate and the therapeutic factor to redirect existing antibodies to tumor cells or infectious organisms or to induce a humoral response against the immunogen can be used. For example, the ligand-immunogen conjugate and the therapeutic factor may be administered as one dose, or they may be administered separately in a multiple dose daily regimen. Furthermore, an alternate regimen, such as 1-3 days per week, may be used as an alternative to daily treatment, and for purposes of defining the present invention such intermittent or alternate daily regimens are considered equivalents of daily treatment and are considered to be within the scope of the present invention. In a preferred embodiment of the invention, the host is treated with multiple injections of the ligand-immunogen conjugate and the therapeutic factor to eliminate a population of pathogenic cells. In one embodiment, the host is injected multiple times (preferably about 2 times up to about 50 times) with the ligand-immunogen conjugate, for example at 12-72 hour intervals or at 48-72 hour intervals. Administering to the patient, after the initial injection, an additional injection of the ligand-immunogen conjugate at an interval of days or months, the additional injection preventing disease recurrence. Alternatively, initial injection of the ligand-immunogen conjugate can prevent disease recurrence.

The therapeutic factor can be administered to the host animal before, after, or simultaneously with the ligand-immunogen conjugate, and the therapeutic factor can be administered as part of the same composition containing the conjugate or as part of a different composition than the ligand-immunogen conjugate. Any such composition containing a therapeutically effective amount of the therapeutic factor may be used in the present invention. In addition, more than one type of ligand-immunogen conjugate may be used. For example, a host animal may be preimmunized with fluorescein isothiocyanate and dinitrophenyl followed by treatment with fluorescein isothiocyanate and dinitrophenyl linked to the same or different ligands in a co-dosing regimen. In the case of chemotherapeutic and antimicrobial agents, the therapeutic factor may be administered in a combination therapy with the ligand-immunogen conjugate at suboptimal doses to avoid the host animal developing resistance to the chemotherapeutic or antimicrobial agent.

The ligand-immunogen conjugate and the therapeutic factor are preferably injected parenterally, which may be intraperitoneal, subcutaneous, intramuscular, intravenous, or intrathecal. The ligand-immunogen conjugate and the therapeutic factor may also be administered using a slow pump. Examples of parenteral dosage forms include aqueous solutions of the active agent in isotonic saline, 5% glucose, or other well-known pharmaceutically acceptable liquid carriers such as liquid alcohols, glycols, esters, and amides. The parenteral dosage form according to the invention may be in the form of a reconstitutable lyophilized formulation comprising the dose of the ligand-immunogen conjugate and the therapeutic factor. In a preferred aspect of the present invention, any number of sustained release dosage forms known in the art may be administered, such as the biodegradable saccharide matrices described in U.S. patent nos. 4,713,249, 5,266,333, and 5,417,982, the disclosures of which are incorporated herein by reference.

The specific implementation mode is as follows:

example 1

Folic acid-fluorescein isothiocyanate conjugates

Effect on survival of mice bearing Lung tumor grafts

6-8 week old (. about.20-22 g) female Balb/c mice were treated with Fluorescein Isothiocyanate (FITC) -labeled Bovine Serum Albumin (BSA) using a commercially available adjuvant (e.g., TiterMax's adjuvant, Freund's adjuvant)^TMGold) subcutaneous immunization at multiple sites. After determining that the anti-FITC antibody titers were high in all mice (as evidenced by ELISA assay results of mouse serum samples), each animal was givenIntraperitoneal injection of 5X 10⁵M109 cells, a homologous lung cancer cell line expressing high levels of folate receptors. Cancer lesions attach (attach) and grow. All animals were injected intraperitoneally with either Phosphate Buffered Saline (PBS) or a specific amount of FITC conjugated with folic acid via a gamma carboxy linked ethylenediamine bridge 4 days and 7 days after transplantation of cancer cells. folate-FITC was injected at concentrations of 0(PBS control), 4.5, 45, 450, and 4500nmole/kg, 8 mice per folate-FITC concentration, for a total of 40 mice. All mice were then given a series of 5 daily injections of 5000IU recombinant human IL-2 (day 8 to day 12) to stimulate the immune system. The efficacy of this immunotherapy was then assessed by monitoring the survival rate of folate-FITC treated mice over time compared to control animals. As shown in figure 1, the mean survival of mice treated with folate-FITC was dose dependent, whereas control mice showed a mean survival of 23 days after tumor transplantation, whereas folate-FITC mice survived longer and longer as the dose of the conjugate increased. Folic acid-FITC at as low as 45nmole/kg promotes long-term survival of mice, while higher doses are proportionally more effective. Although the folate-FITC was found to concentrate in tumors, some folate-FITC was present in kidney tissue (but not at levels in other normal tissues). No nephrotoxicity or toxicity of normal organs was detected by certified veterinary pathologists at necropsy.

Example 2

Imaging of normal and tumor tissue with fluorescein isothiocyanate conjugated folate

The procedure is similar to that described in example 1, except that animals are injected with 24JK-FBP tumor cells, mice are sacrificed shortly after injection of folate-FITC, thin sections of tissue are made, examined by FITC immunofluorescence using focused fluorescence microscopy to localize folate-FITC to specific tissues, including tumor, kidney, liver and muscle tissues. Figure 2 shows phase contrast micrographs and fluorescence micrographs of various thin sections of tissue as controls. folate-FITC was found to localize specifically in tumor tissue and renal proximal tubule cells with a particularly rich folate receptor.

Example 3

Labelled with folic acid or phycoerythrin conjugated with fluorescein isothiocyanate

Goat anti-mouse IgG imaging tumor tissue

The procedure was similar to that described in example 2, except that the tissues were examined by FITC fluorescence (green image) and Phycoerythrin (PE) fluorescence (red image) using M109 cells. For PE fluorescence, the fluorescent label was conjugated to goat anti-mouse IgG antibody for detection of endogenous mouse anti-FITC antibody binding to folate-FITC conjugate, which accumulates on tumor cells. folate-FITC treated tumor tissue was compared to untreated tumor tissue, and both types of samples were also examined by phase contrast microscopy, as described in example 2. FITC fluorescence demonstrated: folate localized to tumor tissue (fig. 3). PE fluorescence proves that: endogenous mouse anti-FITC antibodies conjugated to folate-FITC conjugates localize to tumor cells. Other studies (not shown) demonstrated the lack of binding of such IgG to normal tissues, including the kidney. No antibody was bound to folate-FITC located in kidney tissue due to the fact that: if the folate receptor is on the apical membrane of the kidney proximal tubule cells, the antibody cannot contact this region of the kidney. Phase contrast images (transmitted images) show the morphology of treated and untreated tumor tissue, indicating cell death in the treated sample.

Example 4

Effect of Folic acid fluorescein isothiocyanate conjugates on solid tumor growth

The procedure is similar to that described in example 1, except that each animal is injected subcutaneously in the shoulder 1X 10 after preimmunization with FITC⁶M109 cells (day 0). Tumor(s)Immunization with folate-FITC after cell transplantation included 1500nmol/kg folate-FITC administered at 6 intraperitoneal doses at 48 hour intervals (days 7,9, 11, 13, 15, and 17). The resulting solid tumors of the shoulder were measured and the percentage increase in tumor size was determined. The tumor growth curves shown in FIG. 4 indicate that the growth of solid tumors is significantly inhibited when animals are treated with folate-FITC in combination with IL-2.

Example 5

Effect of cytokine combination therapy

The method is similar to that described in example 1, except that animals are treated with 5 daily injections (days 8 to 12) of 5000IU of recombinant human IL-2 and/or IFN- α (5 daily injections of 2.5X 10) by injecting 2 doses of either folic acid-FITC or aminofluorescein at 1500nmol/kg on days 4 and 7 after tumor cell transplantation⁴U/day), IL-12(5 daily injections of 0.5. mu.g/day) or TNF-alpha (3 daily injections of 2. mu.g/day on days 8, 10 and 12). Furthermore, to reduce the time required to obtain long-term survival data, tumor cells were transplanted intraperitoneally to the vicinity of the liver. Thus, the lifespan of the tumor-bearing mice was generally shortened compared to the mice shown in example 1. The results shown in FIG. 5 demonstrate that IL-2 alone is more effective in promoting long-term survival in animals than combination therapy with IL-2 and IL-12 or with IL-2 and TNF- α. In contrast, combination therapy with IL-2 and IFN- α is more effective in promoting long-term survival than IL-2 alone. Aminofluorescein was injected in combination with various cytokines as a control because the compound did not bind folate and did not re-target the anti-fluorescein antibody to tumor cells.

Example 6

Effect of multiple injections of Folic acid fluorescein isothiocyanate conjugates

The method is similar to that described in example 1 except that animals are injected intraperitoneally at 48 hour intervals, 6 daily injections (days 7,9, 11, 13, 15 and 17 after tumor cell transplantation) of 1500nmol/kg folate-FITC. The results show (FIG. 6) that multiple injections of folate-FITC improved the long-term survival of animals treated with folate-FITC and IL-2 compared to 2 injections of folate-FITC on days 4 and 7 post tumor cell transplantation.

Example 7

Synergistic effects of Folic acid fluorescein isothiocyanate conjugates and IL-2

The method is similar to that described in example 1, except that animals are injected with 1500nmole/kg folate-FITC, and some animals are treated with either folate FITC alone or IL-2 alone. In addition, the tumor cells were transplanted intraperitoneally as described in example 5. This experiment (see FIG. 7) was performed to determine if folate-FITC and IL-2 act synergistically to promote long-term survival of tumor bearing mice. The mean survival time for the control group (n-8) and the group treated with IL-2, folate-FITC or folate-FITC + IL-2 (n-8) was 18 days, 19 days, 22 days and 42 days, respectively. The results shown in FIG. 7 indicate that the ability of folate-FITC and IL-2 to promote long-term survival in tumor-bearing mice is strongly synergistic, with low doses of IL-2 alone having negligible effect on survival in the absence of folate-FITC and only minimal effect of folate-FITC.

Example 8

NK cells are associated with a synergistic effect of fluorescein isothiocyanate conjugate folate and IL-2

The procedure was similar to that described in example 7, except that one group of animals was treated with polyclonal rabbit anti-mouse NK cell antibody (anti-asialo GM 1; Wako Pure chemical industries, Ltd., Richmond, Va.) in combination with folate-FITC and IL-2. Each mouse was injected with a 1: 10 dilution of 0.2ml antibody stock on days 1, 4, 9 and 14 post-tumor transplantation to achieve NK cell depletion. The mean survival time for the control group and the group treated with folate-FITC + IL-2 or folate-FITC + IL-2+ alpha-NK Ab were 18 days, 42 days and 18.5 days, respectively. The results shown in FIG. 8 demonstrate that NK cells mediate a synergistic enhancement of long-term survival of tumor-bearing mice induced by combined treatment with folate-FITC and IL-2.

Example 9

Generation of cellular immunity against M109 tumor cells

The method is similar to that described in example 1, except that the tumor cells are transplanted intraperitoneally at the locations described in example 5, and the animals are injected with PBS (control) or co-injected with folate-FITC (1500nmole/kg), IL-2(250,000 IU/dose), and IFN- α (25,000U/dose) on days 7, 8,9, 11, and 14 after tumor cell transplantation. In addition, by injection of 5X 10 at day 62 post initial tumor cell transplantation⁵M109 cells injected at 1.5X 10 day 96 post primary tumor cell transplantation⁶M109 cells, or 2.5X 10 injection on day 127 after initial tumor cell transplantation⁵Line 1 cells, a Balb/c spontaneous lung carcinoma, challenged animals.

As shown in FIG. 9, 5X 10 injections were administered⁵The mean survival time of control mice of M109 cells was 18.5 days. Injection of 1.5X 10⁶The mean survival time of control mice with M109 cells was 18 days. Injection of 2.5X 10⁵The mean survival time of control mice of Line 1 cells was 23.5 days. Injection of 5X 10⁵M109 cells, treatment with folate-FITC in combination with IL-2 and IFN- α, 5X 10 on day 62⁵Challenge with M109 cells, 1.5X 10 on day 96⁶Challenge with M109 cells and 2.5X 10 on day 127⁵The mean survival time of the Line 1 cell challenged mice was over 192 days.

The results shown in FIG. 9 demonstrate that long-lasting cell type specific cellular immunity is generated in animals treated with folate-FITC in combination with IL-2 and IFN- α. This long-lasting immunity protects animals transplanted with M109 cells and receiving folate-targeted immunotherapy from disease recurrence upon challenge by subsequent injection of M109 cells. After the last challenge with Line 1 cells, the survival time in these animals was likely due to the presence of folate receptors at levels lower on Line 1 cells than on M109 cells, and due to the presence of shared tumor antigens between M109 cells and Line 1 cells, elicited M109-specific cellular immune responses that were capable of cross-reacting with Line 1 cells.

Example 10

Effect of IL-2 dose on survival of mice treated with folate-fluorescein isothiocyanate conjugates

The method is similar to that described in example 1, except that the tumor cells are transplanted intraperitoneally at the locations described in example 5, and the animals are treated with PBS (control) on days 7, 8,9, 11, and 14 after tumor cell transplantation, or the animals are co-injected with folate-FITC (1500nmole/kg) and 5X 10³IU(1×)、0.5×10⁵IU(10×)、2.5×10⁵IU (50X) or 5X 10⁵IU (100X) dose of IL-2. In addition, animals were keyhole limpet labeled with FITCHemocyanin (KLH) was used instead of FITC-labeled BSA. As shown in FIG. 10, the mean survival time of mice transplanted with M109 cells and treated with folate-FITC increased when the dose of IL-2 was increased to greater than 5X 10³IU IL-2 dose was increased. In contrast, there was no substantial difference between the mean survival time of control mice (mice injected with M109 cells and treated with PBS) and mice treated with IL-2 alone.

Example 11

IFN-alpha enhancement of survival of mice treated with folate-fluorescein isothiocyanate conjugate and IL-2

The method is similar to that described in example 1, except that the tumor cells are transplanted intraperitoneally at the locations described in example 5, and the animals are treated with PBS (control) on days 7, 8,9, 11, and 14 after tumor cell transplantation, or the animals are co-injected with folate-FITC (1500nmole/kg) and IL-2(5000 IU/dose) or folate-FITC (1500nmole/kg), IL-2(5000 IU/dose), and IFN- α (25,000U/dose). Another group of mice was co-injected with folate-FITC, IL-2, and IFN- α, but the animals were not preimmunized with BSA-FITC. FIG. 11 shows that the mean survival time for control mice treated with PBS was 18.5 days, for mice co-injected with folate-FITC and IL-2 was 20.5 days, for mice co-injected with folate-FITC, IL-2, and IFN- α was over 60 days, and for mice co-injected with folate-FITC, IL-2, and IFN- α, but not pre-immunized, was 24.3 days. The mean survival time of mice injected with folate-FITC and IL-2 was not substantially different from that of control mice, since the mice were injected with 5000IU of IL-2, as described in example 10, with the regimen on days 7, 8,9, 11 and 14, increasing the mean survival time of mice treated with folate-FITC required doses of IL-2 higher than 5000 IU. The results shown in FIG. 11 indicate that IFN- α further enhances the increase in mean survival time that occurs as a result of treatment of mice transplanted with tumor cells with folate-FITC and IL-2.

Example 12

CD8⁺Effect of T cell depletion on folate-targeted immunotherapy

The method is similar to that described in example 1, except that the tumor cells are transplanted intraperitoneally at the locations described in example 5, and the animals are treated with PBS (control) on days 7, 8,9, 11, and 14 after tumor cell transplantation, or the animals are co-injected with folate-FITC (1500nmole/kg), IL-2(5000 IU/dose), and IFN- α (25,000U/dose). Another group of mice was co-injected with aminofluorescein (1500nmole/kg), IL-2 and IFN- α or with folate-FITC, IL-2, IFN- α and anti-CD 8⁺T cell antibodies (in ascites form and given on days 2, 3, 7, 11 and 15). As shown in FIG. 12, anti-CD 8⁺T cell antibodies inhibited the increase in mean survival time of mice treated with folate-FITC, IL-2, and IFN- α, indicating CD8⁺T cellsPlays a role in activating cellular immune responses by folate-targeted immunotherapy. Aminofluorescein was injected as well as the IL-2, IFN-. alpha.cytokine combination as a control, since this compound did not bind folic acid and did not redirect anti-fluorescein antibodies to tumor cells. FIG. 12 shows that the efficacy of aminofluorescein together with IL-2 and IFN- α is much lower than folate-FITC, IL-2 and IFN- α in increasing the mean survival time of mice engrafted with M109 cells.

Example 13

Increased Effect of GM-CSF on IL-2 and IFN-alpha enhanced folate-targeted immunotherapy

The method is similar to that described in example 1, except that the tumor cells are transplanted intraperitoneally at the locations described in example 5. In addition, as shown in FIG. 13, animals were injected with various cytokines including IL-2(5000 IU/dose), IFN- α (25,000U/dose), and GM-CSF (3000 IU/dose). After transplantation of M109 cells and subsequent injection of 2 doses of 1500nmole/kg folate-FITC on days 4 and 7, the cytokines were co-injected in a series of 5 daily injections on days 8 to 12. The results shown in FIG. 13 indicate that the mean survival time for mice treated with PBS was 19 days, for mice injected with IL-2, IFN- α and GM-CSF but no folate-FITC was 22 days, for mice injected with folate-FITC, IL-2 and IFN- α was 38 days, and for mice injected with folate-FITC, IL-2, IFN- α and GM-CSF was over 57.5 days. The results indicate that GM-CSF further enhances folate-targeted tumor cell killing in mice also treated with IL-2 and IFN- α. The mean survival time of mice injected with PBS, IL-2, IFN- α and GM-CSF was not significantly different from control mice, indicating the importance of targeting tumor-specific immune responses with folate-FITC.

Example 14

Effect of IFN-alpha dose on survival of mice treated with folate-fluorescein isothiocyanate conjugates

The method is similar to that described in example 1, except that the tumor cells are transplanted intraperitoneally at the locations described in example 5, and the animals are treated with PBS (control), or the animals are co-injected with folate-FITC (1500nmole/kg) and 1.5X 10⁵IU/dose (6X), 7.5X 10⁴IU/dose (3X), 2.5X 10⁴IU/dose (1X) or and 7.5X 10³IU (0.3X) dose of IFN-alpha. In addition, animals were keyhole limpet labeled with FITCHemocyanin (KLH) was immunized instead of FITC-labeled BSA, and animals were injected with folate-FITC and IFN- α on days 7, 8,9, 11, and 14 after tumor cell transplantation. As shown in FIG. 14, the mean survival time of mice transplanted with M109 cells and treated with folate-FITC was increased when the IFN- α dose was increased to above 0.8X 10⁴IU/dose of IFN-alpha dose is increased.

Example 15

Effect of dinitrophenyl as immunogen on folate-targeted immunotherapy

The method is similar to the method described in example 1, except that the tumor cells are transplanted intraperitoneally at the locations described in example 5, and the animals are treated with PBS (control) on days 7, 8,9, 11, and 14 after tumor cell transplantation, or the animals are co-injected with Dinitrophenyl (DNP) (1500nmole/kg), IL-2(5000 IU/dose/day), and IFN-alpha (2.5X 10)⁴Unit/day) or co-injection of folate-Dinitrophenyl (DNP) (1500nmole/kg), IL-2(5000 IU/dose/day) and IFN-alpha (2.5X 10)⁴Unit/day). In addition, animals were keyhole marked with DNPHemocyanin (KLH) immunization. As shown in FIG. 15, mean survival time was increased in mice treated with folate-DNP, IL-2 and IFN- α compared to control mice (treated with PBS) or mice treated with DNP, IL-2 and IFN- α. Thus, DNP is alsoAn effective immunogen for folic acid targeted immunotherapy.

Example 16

Synergistic effects of Folic acid fluorescein isothiocyanate conjugates and IFN-alpha

The method was similar to that described in example 1, except that the tumor cells were transplanted intraperitoneally at the locations described in example 5, and animals were treated with PBS (control), IFN- α alone (7.5X 10) on days 7, 8,9, 11, and 14 after tumor cell transplantation⁴Unit/dose) or folic acid FITC alone (1500nmole/kg), or animals co-injected with folic acid FITC (1500nmole/kg) and IFN-alpha (7.5X 10)⁴Units/dose). In addition, animals (5 mice per group) were keyhole limpet labeled with FITCHemocyanin (KLH) was used instead of FITC-labeled BSA. As shown in FIG. 16, the mean survival time of the groups treated with PBS (control), IFN-. alpha., folate-FITC, or folate-FITC + IFN-. alpha.was 17 days, 23 days, and 33 days, respectively. These results indicate that IFN- α, like IL-2, acts synergistically with folate-FITC to promote long-term survival in tumor bearing mice.

Example 17

Dinitrophenyl as immunogen and high concentration of cytokine

Effect on Long-term survival of mice

The method is similar to that described in example 1, except that the tumor cells are transplanted intraperitoneally at the sites described in example 5, and the animals are treated with PBS (control) on days 7, 8,9, 11, and 14 after tumor cell transplantation, or the animals are co-injected with PBS, IL-2 (2.5X 10)⁵Unit/day) and IFN-. alpha.s (7.5X 10)⁴Unit/day) or co-injection of folic acid-Dinitrophenyl (DNP) (1500nmole/kg), IL-2 (2.5X 10)⁵Unit/day) and IFN-. alpha.s (7.5X 10)⁴Unit/day). In addition, animals were keyhole marked with DNPHemocyanin (KLH) immunization. As shown in FIG. 17, the mean survival time of mice treated with folate-DNP, IL-2 and IFN- α was increased compared to control mice (treated with PBS) or mice treated with PBS, IL-2 and IFN- α. With folic acid-DNP, IL-2 and IFN-alpha (IL-2 and IFN-alpha at concentrations of 2.5X 10, respectively)⁵Unit/day and 7.5X 10⁴Unit/day) treated mice were completely cured.

Claims (45)

1. A method of enhancing an endogenous immune response-mediated specific elimination of a population of pathogenic cells in a host animal harboring said population, wherein members of said cell population have binding sites accessible to ligands, said method comprising the steps of: administering to said host

A ligand-immunogen conjugate composition comprising a complex of said ligand and an immunogen wherein said immunogen is known to be recognized by endogenous or exogenous antibodies in said host, or said immunogen is known to be recognized directly by immune cells in said host; and

at least one additional composition comprising a therapeutic factor selected from the group consisting of a cell killing agent, a tumor penetration enhancer, a chemotherapeutic agent, an antimicrobial agent, a cytotoxic immune cell, and a compound capable of stimulating an endogenous immune response, wherein the compound is not bound to the ligand-immunogen conjugate.

2. The method of claim 1, wherein said population of pathogenic cells is a population of cancer cells.

3. The method of claim 2, wherein the population of cancer cells is tumorigenic.

4. The method of claim 1, wherein the population of pathogenic cells is an exogenous pathogen or an endogenous population of cells harboring an exogenous pathogen.

5. The method of claim 4, wherein said exogenous pathogen is selected from the group consisting of a bacterium, a fungus, a virus, a mycoplasma, and a parasite.

6. The method of claim 1, wherein the ligand is a vitamin capable of specifically binding to a cell membrane receptor.

7. The method of claim 6, wherein said ligand is selected from the group consisting of folate and other folate receptor binding ligands.

8. The method of claim 1, wherein the ligand is chemically complexed with the immunogen by a linkage comprising a covalent bond, an ionic bond, or a hydrogen bond (complex).

9. The method of claim 8, wherein said ligand is a folic acid analog having one glutamyl moiety covalently linked to said immunogen only through the glutamyl γ -carboxy moiety of said ligand.

10. The method of claim 8, wherein said ligand is a folate having a glutamyl moiety covalently linked to said immunogen only through the glutamyl α -carboxyl moiety of said ligand.

11. The method of claim 9 or 10, wherein the covalent linkage between the immunogen and the ligand is either a direct covalent linkage to the immunogen or a covalent linkage through a divalent linker.

12. The method of claim 1, wherein the ligand is a small organic molecule capable of binding a receptor, and wherein the receptor is preferentially expressed, uniquely expressed, or overexpressed on the surface of the population of pathogenic cells.

13. The method of claim 12, wherein the small organic molecule is an antimicrobial agent.

14. The method of claim 1, wherein the ligand is a β -lactam antibiotic.

15. The method of claim 1, wherein the ligand binding site is an antigen that is preferentially expressed, uniquely expressed, or overexpressed on metastatic cancer cells.

16. The method of claim 15 wherein said ligand binding site is EphA 2.

17. The method of claim 1 wherein said immunogen is an organic molecule having a molecular weight of less than 20,000 daltons.

18. The method of claim 17, wherein the organic molecule is fluorescein or dinitrophenyl.

19. The method of claim 1 wherein the immunogen is alpha-galactosyl.

20. The method of claim 1, wherein the antibody is foreign to the host and the antibody is co-administered with the conjugate composition.

21. The method of claim 1, wherein the therapeutic factor comprises a cytokine.

22. The method of claim 21, wherein the therapeutic factor comprises IL-2, IL-12, IL-15, or a combination thereof.

23. The method of claim 21, wherein the therapeutic factor comprises a combination of IL-2, IL-12, IL-15, or a combination thereof and IFN- α or IFN- γ.

24. The method of claim 21, wherein the therapeutic factor comprises a combination of IL-2, IL-12, IL-15, or a combination thereof, and IFN- α or IFN- γ, or a combination thereof, and GM-CSF.

25. The method of claim 21, wherein the therapeutic factor comprises at least one NK cell or T cell stimulator.

26. The method of claim 1, wherein the ligand-immunogen conjugate composition is administered in multiple injections.

27. The method of claim 1, wherein the host animal has been previously naturally exposed to the immunogen such that the host animal has had an existing immunity to the immunogen as evidenced by the presence of endogenous antibodies to the immunogen.

28. The method of claim 1, wherein the host animal has been previously exposed to the immunogen by an unnatural process, resulting in eliciting an immune response in the host animal against the immunogen.

29. The method of claim 28, wherein the non-native process that results in eliciting an immune response in the animal is vaccination.

30. The method of claim 28, wherein the non-native process that results in eliciting the immune response is active immunization.

31. The method of claim 1, wherein the endogenous immune response comprises a humoral immune response.

32. The method of claim 31, wherein said humoral response is an acquired immune response.

33. The method of claim 31, wherein said humoral response is an innate immune response.

34. The method of claim 32, wherein said adaptive response is induced by administering a vaccine composition to said host animal.

35. The method of claim 1, wherein the endogenous immune response comprises a cell-mediated immune response.

36. The method of claim 1, wherein the endogenous immune response comprises a humoral immune response and a cell-mediated immune response.

37. A method of enhancing an endogenous immune response-mediated specific elimination of a population of pathogenic cells in a host animal harboring said population, wherein said population expresses a binding site for a ligand, said method comprising the steps of:

administering to said host a composition comprising a complex of said ligand and an immunogen;

administering to said host antibodies directed against said immunogen; and

administering to said host at least one additional therapeutic factor selected from the group consisting of a cell killing agent, a tumor penetration enhancer, a chemotherapeutic agent, an antimicrobial agent, a cytotoxic immune cell, and a stimulator of an endogenous immune response not associated with said ligand-immunogen complex.

38. A method of enhancing an endogenous immune response-mediated specific elimination of a population of pathogenic cells in a host animal harboring said population, wherein said population preferentially expresses, uniquely expresses, or overexpresses a folate receptor, said method comprising the steps of: administering to said host

A composition comprising a covalently linked conjugate of an immunogen and a ligand, wherein the immunogen is known to be recognized by endogenous or exogenous antibodies in the host, or the immunogen is known to be recognized directly by immune cells in the host;

the ligand comprises folic acid or a folic acid analogue having a glutamyl group, wherein the covalent attachment to the immunogen is via the gamma-carboxyl group of the glutamyl group only.

39. A method of enhancing an endogenous immune response-mediated specific elimination of a population of pathogenic cells in a host animal harboring said population, wherein said population preferentially expresses, uniquely expresses, or overexpresses a binding site for a folate receptor, said method comprising the steps of: administering to said host

A composition comprising a covalently linked conjugate of an immunogen and a ligand, wherein the immunogen is known to be recognized by endogenous or exogenous antibodies in the host, or the immunogen is known to be recognized directly by immune cells in the host;

the ligand comprises folic acid or a folic acid analogue having a glutamyl group, wherein the covalent attachment to the immunogen is via the α -carboxyl group of the glutamyl group only.

40. A method of enhancing an endogenous immune response-mediated specific elimination of a population of pathogenic cells in a host animal harboring said population, wherein said population preferentially expresses, uniquely expresses, or overexpresses a binding site for a folate receptor, said method comprising the steps of: administering to said host

A composition, comprising: a covalently linked conjugate of an immunogen, wherein the immunogen is known to be recognized by endogenous or exogenous antibodies in the host, or is known to be recognized directly by immune cells in the host;

a ligand comprising folic acid or a folic acid analogue having a glutamyl group, wherein said covalent attachment is via only the gamma-carboxyl group of said glutamyl group; and

at least one additional composition comprising a therapeutic factor selected from the group consisting of a cell killing agent, a tumor penetration enhancer, a chemotherapeutic agent, an antimicrobial agent, a cytotoxic immune cell, and a compound capable of stimulating an endogenous immune response, wherein the compound is not bound to the ligand-immunogen conjugate.

41. A method of enhancing an endogenous immune response-mediated specific elimination of a population of pathogenic cells in a host animal harboring said population, wherein said population preferentially expresses, uniquely expresses, or overexpresses a folate receptor, said method comprising the steps of: administering to said host

A composition, comprising: a covalently linked conjugate of an immunogen, wherein the immunogen is known to be recognized by endogenous or exogenous antibodies in the host, or is known to be recognized directly by immune cells in the host;

a ligand comprising folic acid or a folic acid analogue having a glutamyl group, wherein said covalent attachment is via only the a-carboxyl group of said glutamyl group; and

at least one additional composition comprising a therapeutic factor selected from the group consisting of a cell killing agent, a tumor penetration enhancer, a chemotherapeutic agent, an antimicrobial agent, a cytotoxic immune cell, and a compound capable of stimulating an endogenous immune response, wherein the compound is not bound to the ligand-immunogen conjugate.

42. A pharmaceutical composition comprising: a therapeutically effective amount of a ligand-immunogen conjugate capable of specifically binding to a population of pathogenic cells in a host animal to specifically eliminate said cells directly by an acquired or innate immune response, a co-administered antibody, or by immune cells in the host; a therapeutic factor selected from the group consisting of a cell killing agent, a tumor penetration enhancer, a chemotherapeutic agent, an antimicrobial agent, and a compound capable of stimulating an endogenous immune response, wherein the compound is not bound to the ligand-immunogen conjugate; and a pharmaceutically acceptable carrier therefor.

43. The pharmaceutical composition of claim 42, which is in a parenteral sustained release dosage form.

44. The pharmaceutical composition of claim 42, wherein said therapeutic factor is an immunostimulant.

45. The pharmaceutical composition of claim 44, wherein the immunostimulatory substance comprises a compound or combination thereof selected from the group consisting of: IL-2, IL-12, IL-15, IFN-alpha, IFN-gamma and GM-CSF.