Title:  Cellular vaccines and immunotherapeutics and methods for their preparation

United States Patent:  6,805,869

Issued:  October 19, 2004

Inventors:  Guo; Yajun (San Diego, CA)

Assignee:  Shanghai CP Guojian Pharmaceutical Co., Ltd. (Shanghai, CN)

Appl. No.:  216604

Filed:  December 17, 1998

Abstract

The present invention provides a method for enhancing the immunogenicity of weakly immunogenic or non-immunogenic cells, resulting in a cellular vaccine that can stimulate T cell activation, which in turn leads to an effective immune response. The cellular vaccines of the present invention are useful for the prevention and treatment of diseases which develop and/or persist by escaping the immune response triggered by T cell activation. Such diseases include, for example, all cancers, natural and induced immune deficiency states, and diseases caused by infections with a variety of pathogens.

SUMMARY OF THE INVENTION

The present invention features immunogenic tumor cells and other immunogenic autologous cells, convenient methods of making such immunogenic cells, methods of using such immunogenic cells to activate or enhance immune response against diseased cells with minimum effect on normal or healthy cells, and methods of avoiding the negative T cell signaling pathway.

The present invention provides a method for enhancing the immunogenicity of weakly-immunogenic or non-immunogenic cells, resulting in a cellular vaccine that can stimulate T cell activation, which in turn leads to an effective immune response against diseased cells. The cellular vaccines of the present invention can be used as vaccines to prevent diseases and as immunotherapeutics to treat diseases. The starting materials for the cellular vaccine can be a target diseased cell (e.g., autologous or in vivo diseased cells and in vitro transformed cell lines), or an antigen presenting cell presenting one or more antigens associated with a disease (e.g., dendritic cells, macrophages, B cells, and other cells fused with diseased cell, pulsed with antigens or transfected with antigen expressing nucleic acid).

In summary, the method of the invention involves the steps of (1) treating weakly- or non-immunogenic autologous cells (target cells) in order to amplify primary and costimulatory T cell activation signals in the cells, and (2) attaching to the treated cells a substance capable of binding to one or more antigens on the treated cells and to one or more T cell activation costimulatory molecules on the surface of T cells (such as CD28), thereby providing the treated cells with the capacity to physically link to T cells and to activate the costimulatory signal. Such substances include, but are not limited to, bispecific monoclonal antibodies (Bi-MAbs) targeted to antigen on the treated cells and to CD28 and/or other costimulatory molecules on T cells. The first step may be skipped when the autologous cell is attached with (1) a bridge molecule with two or more binding sites for T cell activation costimulatory molecules on the surface of T cells, or (2) two or more bridge molecules each with one or more binding sites for T cell activation costimulatory molecules on the surface of T cells. The fist step may also be skipped when the target cells are antigen presenting cells presenting one or more antigens associated with a disease.

Once the primary and/or costimulatory T cell activation signals in the target diseased cells have been amplified by cytokines or other means and the bridge molecules have been attached to the target diseased cells, the cytokines and the bridge molecules not attached to the target diseased cells may be removed from the immunogenic composition before the target diseased cells are administered to a patient. This additional step minimizes adverse effects associated with administering cytokines to a patient. It also minimizes the risk associated with allowing bridge molecules not attached to a target diseased cell into a patient, an event which may cause unwanted immune response against normal or healthy cells.

The first step of the method up-regulates antigen processing capacity within the treated cells and amplifies the expression of cell surface molecules involved in T cell activation. The second step provides the treated cells with a means to physically bridge to T cells via CD28 and/or other costimulatory molecules, thereby providing optimal conditions for stimulating T cell activation.

Thus, in a first aspect, this invention features an immunogenic composition for administration to a patient mammal (including a human) having target diseased cells. The immunogenic composition contains an autologous target diseased cell which differs from the diseased cells in the patient in that it processes and presents antigens characteristic of the diseased cells more effectively. For example, the autologous target diseased cell expresses one or more primary (e.g., MHC) and/or costimulatory (e.g., B7-1 and B7-2) T cell activation molecules at a higher level (e.g., 50% higher, preferably 2 folds higher, more preferably 10 folds higher). As described below, there are different ways of enhancing the expression level of the primary and/or costimulatory T cell activation molecules.

In addition, the autologous target diseased cell has attached thereto one or more bridge molecules. Each bridge molecule has one or more binding sites for one or more costimulatory molecules on the surface of effector cells, which include, but are not limited to, T cells, NK cells, macrophages, LAK cells, B cells, and other white blood cells. Preferably, though not required, the bridge molecules have one or more binding sites for one or more antigens on the surface of the target diseased cell and are attached to the target diseased cells at the cell surface antigens. In another preferred embodiment, substantially all (e.g., >80%, preferably >90%, more preferably >95%) the bridge molecules in the immunogenic composition are attached to the autologous target diseased cells so that the composition is substantially free of bridge molecules not attached to a target diseased cell. In a further preferred embodiment, the immunogenic composition contains a pharmaceutically effective amount of the target diseased cells with bridge molecules attached thereto.

By "immunogenic" is meant the ability to activate the response of the whole or part of the immune system of a mammal, especially the response of T cells.

By "autologous" is meant that the target diseased cell is from the patient mammal, or from another patient having a common major histocompatibility phenotype. An autologous target cell may be obtained from the patient mammal or another source sharing the same MHC with methods known to those skilled in the art. Once taken from a patient, an autologous cell may be modified, transfected, and treated by methods described herein.

By "target diseased cell" is meant a cell causing, propagating, aggravating or contributing to a disease in a patient mammal. Target diseased cells include, but are not limited to, tumor cells (including unmodified tumor cells, tumor cells modified with different approaches, and primary culture). The sources of tumor cells include, but are not limited to, liver cancer, hepatocellular carcinoma, lung cancer, gastric cancer, colorectal carcinoma, renal carcinoma, head and neck cancers, sarcoma, lymphoma, leukemia, brain tumors, osterosarcoma, bladder carcinoma, myloma, melanoma, breast cancer, prostate cancer, ovarian cancer, and pancreas carcinoma.

Target diseased cells may also be cells infected with prions (which cause Mad Cow diseases among others), viruses, bacteria, fungi, protozoa or other parasites (e.g. worms).

Viruses include those described or referred to in Fields Virology Second Edition, 1990, Raven Press, New York, incorporated by reference herein. Examples include, but are not limited to, herpes virus, rhinoviruses, hepatitis virus (type A, B, C and D), HIV, EBV, HPV, and HLV.

Bacteria include those described or referred to in Bergey's Manual of Determinative Bacteriology Ninth Edition, 1994, Williams and Wilkins, incorporated by reference herein. Examples include, but are not limited to, gram positive and negative bacteria, streptococci, pseudomonas and enterococci, Mycobacterium tuberculosis, Aeromonas hydrophilia, Aeromonas caviae, Aeromonas sobria, Streptococcus uberis, Enterococcus faecium, Enterococcus faecalis, Bacillus sphaericus, Pseudomonas fluorescens, Pseudomonas putida, Serratia liquefaciens, Lactococcus lactis, Xanthomonas maltophilia, Staphylococcus simulans, Staphylococcus hominis, Streptococcus constellatus, Streptococcus anginosus, Escherichia coli, Staphylococcus aureus, Mycobacterium fortuitum, and Klebsiella pneumonia.

Primary T cell activation molecules include MHC class I, MHC class II and other molecules associated with antigen processing and/or presentation. Costimulatory T cell activation molecules include ICAM-1, ICAM-2, ICAM-3, LFA-1, LFA-2, VLA-1, VCAM-1, 4-1-BB, B7-1, B7-2, and other cell adhesion proteins and other cell surface proteins which can activate T cell costimulatory pathways through T cell surface proteins.

By "bridge molecule" is meant a molecule or substance which can bring two or more cells together by attaching to the cells with its binding sites. Preferably, a bridge molecule can bring an autologous target diseased cell together with an effector cell and deliver a signal to the effector cell to activate or enhance the effector cell's immune response against the target. A bridge molecule has one or more binding sites for stimulatory and/or costimulatory molecules on the effector cells. These binding sites can be designed to activate a positive regulator of T cell activation (e.g., CD28, 4-1BB) but avoid stimulating a negative regulator of T cell activation (e.g., CTLA-4). The binding sites can also be designed to blockade a negative regulator of T cell activation (see Leach et al., Science 271:1734-1736, 1996). A bridge molecule may also have one or more binding sites for antigens on the surface of the target diseased cell. Bridge molecules include, but are not limited to, bispecific monoclonal antibodies, fusion proteins, organic polymers, and hybrids of chemical and biochemical materials. The antibodies described or disclosed in U.S. Pat. Nos. 5,601,819, 5,637,481, 5,635,602, 5,635,600, 5,591,828, 5,292,668 and 5,582,996 are incorporated by reference herein.

The antigen on the target cell serving as an anchor for the bridge molecule need not be unique to the target cell when the bridge molecule is attached to the target cell in vitro. Any molecule on the target cell surface can be used to anchor the bridge molecule, including, but not limited to, proteins, glycoproteins, lipids, glycolipids, phospholipids, lipid aggregates, steroids, and carbohydrate groups such as disaccharides, oligosaccharides and polysaccharides (see "Molecular Biology of The Cell," pp47-58, pp276-337, Second Edition, published by Garland Publishing, Inc. NY & London). Examples include transferrin receptor, Low Density Lipoprotein (LDL) receptor, gp55, gp95, gpl 15, gp210, CD44, ICAM-1, ICAM-2, collagen and fibronectin receptor, transferrin receptors, Fc receptor, and cytokine receptors.

Costimulatory molecules on the surface of effector cells may be antigens, fatty acids, lipids, steroids and sugars that can stimulate or costimulate these effector cells' functions to destroy the target cells. Costimulatory molecules include, but are not limited to, CD1 a, CD1b, CD1c, CD2, CD2R, CD3, CD4, CD5, CD6, CD7, CD8, CD9, CD10, CD11a, CD11b, CD11c, CDw12, CD13, CD14, CD15, CD15s, CD16a, CD16b, CDw17, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD32, CD33, CD34, CD35, CD36, CD37, CD38, CD39, CD40, CD41, CD42a, CD42b, CD42c, CD42d, CD43, CD44, CD44R, CD45, CD45RA, CD45RB, CD45RO, CD46, CD47, CD48, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD50, CD51, CD51/61 complex, CD52, CD53, CD54, CD55, CD56, CD57, CD58, CD59, CDw60, CD61, CD62E, CD62L, CD62P, CD63, CD64, CDw65, CD66a, CD66b, CD66c, CD66d, CD66e, CD67, CD68, CD69, CD70, CD71, CD72, CD73, CD74, CDw75, CDw76, CD77, CDw78, CD79a, CD80, CD81, CD82, CD83, CDw84, CD85, CD86, CD87, CD88, CD89, CDw90, CD91, CDw92, CD93, CD94, CD95, CD96, CD97, CD98, CD99, CD99R, CD100, CDw101, CD102, CD103, CD104, CD105, CD106, CD107a, CD107b, CDw108, CDw109, CD110-CD114, CD115, CDw116, CD117, CD118*, CD119, CD120a, CD120b, CDw121a, CDw121b, CD122, CD123*, CDw124, CD125*, CD126, CDw127, CDw128, CD129, CDw130, LFA-1, LFA-2, LFA-3, VLA-1, VCAM-1, VCAM-2, 4-1BB, cytokine and chemokin receptors. In a preferred embodiment, the bridge molecule has a binding site for CD28 or 4-1BB on the surface of T cells.

By "pharmaceutically effective" is meant the ability to cure, reduce or prevent one or more clinical symptoms caused by or associated with the diseased cells in the patient mammal, including, but not limited to, uncontrolled cell proliferation, bacteria infection, and virus infection.

The immunogenic composition may be isolated, enriched or purified for administration to a patient.

By "isolated" in reference to the immunogenic composition is meant that the autologous target diseased cell is isolated from a natural source. Use of the term "isolated" indicates that one or more naturally occurring materials have been removed from the normal environment. Thus, the target diseased cell may be placed in a different cellular environment or in a solution free of other cells. The term does not imply that the target diseased cell is the only cell present, but does indicate that it is the predominate cell present (at least 20-50% more than any other cells) and is essentially free (about 90% pure at least) of other tissues naturally associated with it in the body of the patient. In a preferred embodiment, the composition is substantially free of effector cells such as T cells. In another preferred embodiment, the composition is substantially free of bridge molecules not attached to a target diseased cell. In a third preferred embodiment, the composition is substantially free of cytokines outside of the target diseased cell.

By "enriched" in reference to the immunogenic composition is meant that the autologous target diseased cell constitutes a significantly higher fraction (2-5 fold) of the total cells in the composition than in the diseased tissue in the patient's body. This could be caused by a person by preferential reduction in the amount of other cells present, or by a preferential increase in the amount of the specific target diseased cells, or by a combination of the two. However, it should be noted that enriched does not imply that there are no other cells present, just that the relative amount of the cell of interest has been significantly increased in a useful manner. The term "significantly" here is used to indicate that the level of increase is useful to the person making such an increase, and generally means an increase relative to other cells of about at least 2 fold, more preferably at least 5 to 10 fold or even more.

By "purified" in reference to the immunogenic composition does not require absolute purity (such as a homogeneous preparation); instead, it represents an indication that the target diseased cell is relatively purer than in the natural environment. The target diseased cells could be obtained directly from the patient or from cell culture, with or without modifications. Purification of at least one order of magnitude, preferably two or three orders, and more preferably four or five orders of magnitude is expressly contemplated. In a preferred embodiment, the composition is substantially free of effector cells such as T cells.

The immunogenic composition may contain a pharmaceutically suitable carrier or excipient. Techniques for formulation and administration may be found in Remington's Pharmaceutical Sciences, 18th ed., Mack Publishing Co., Easton, Pa. (1990). The immunogenic composition may be administered to a patient systemically, e.g., by intravenous infusion or subcutaneous injection. A composition of the invention may be administered as a unit dose to a patient mammal, each unit containing a predetermined quantity (e.g., about 1x10⁵ to about 1x10¹⁰, preferably about 1x10⁶ to about 1x10⁹, and more preferably about 1x10⁷ to about 1x10⁸) of armed and/or activated autologous target diseased cells calculated to produce the desired therapeutic effect in association with the physiologically tolerable aqueous medium as diluent.

The expression of primary and costimulatory T cell activation molecules may be enhanced by various means, for example, in vitro, ex vivo or in vivo treatment of target cells with cytokines or other factors capable of inducing the desired amplification; and in vitro and in vivo transfer to the target cells of MHC genes, adhesion molecule genes, cytokine genes, and/or their respective transcription activators or enhancers. Cytokines include those described or referred to in The Cytokine Handbook, Thomson, A., (ed.), 1994, Academic Press, San Diego, incorporated by reference herein. Examples include, but are not limited to, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, G-CSF, GM-CSF, inteferons (e.g., IFN .alpha., .beta., and .gamma.), tumor necrosis factors (e.g., INF.alpha., and .beta.) and other chemokines and lymphokines. In a preferred embodiment, IFN.gamma. and TNF.alpha. are used either alone or in combination to enhance the expression of primary and costimulatory T cell activation molecules in autologous target diseased cells.

The bridge molecule may be attached to the target cells by various means, for example, in vitro, ex vivo or in vivo treatment of target cells with the bridge molecule. When a target diseased cell coated with bridge molecules is administered into a patient, it will bind to costimulatory molecules on the surface of the effector cells. The more densely the target diseased cell is coated with bridge molecules, the more effector cells it will be able to bind. In addition, the more binding sites a bridge molecule has for the costimulatory molecules, the more effector cells it will be able to bind.

In that regard, Applicant has found that a cellular vaccine may be prepared without the need of cytokine treatment (to increase the levels of primary and costimulatory T cell activation molecules) when a plurality of bridge molecules are attached to a target cell with binding sites for two or more different costimulatory molecules on the surface of T cells (e.g., CD3, CD28, and 4-1BB). Individual bridge molecules may be attached to different anchor molecules on the surface of the target diseased cell. An individual bridge molecule may also have two or more binding sites for two or more different costimulatory molecules on the surface of T cells.

Thus, in a second aspect, this invention features an immunogenic composition containing an autologous target diseased cell having attached thereto (a) a bridge molecule which has two or more binding sites for two or more different effector cells, (b) a bridge molecule which has two or more binding sites for two or more different costimulatory molecules on the surface of effector cells, (c) two or more bridge molecules each containing a binding site for a different effector cell, (d) two or more bridge molecules each containing a binding site for a different costimulatory molecule on the surface of effector cells, (d) two or more bridge molecules each attached to a different antigen on the target cells, or (e) a combination of two or more of the above.

A pharmaceutically effective amount of an immunogenic composition of this invention may be complemented by a pharmaceutically acceptable carrier before administration to a patient mammal.

Alternatively, a patient may be administered with a pharmaceutical composition containing (1) a pharmaceutically effective amount of a cytokine capable of increasing the level of one or more primary and costimulatory T cell activation molecules in tumor cells, (2) a pharmaceutically effective amount of a bridge molecule containing a binding site for an antigen on the surface of the tumor cells and a binding site for a costimulatory molecules on the surface of T cells, and (3) a pharmaceutically acceptable carrier.

In treating a patient, the autologous target cell may be treated with cytokines or other means of increasing primary and costimulatory T cell activation molecules in vitro before the target cell is administered to the patient. Alternatively, the cytokines may be administered to the patient to increase primary and costimulatory T cell activation molecules in vivo.

In a third aspect, this invention features a method of generating cytotoxic leukocytes against diseased cells in a patient mammal by contacting a population of effector cells (e.g., white blood cells) in vitro with immunogenic compositions described above for a time period sufficient to react with the immunogenic compositions and collecting the treated effector cell population. The cytotoxic leukocytes so generated can then be administered to a patient to treat or prevent diseases. This adoptive immunotherapy can be used alone or in combination with vaccination to treat or prevent diseases.

The method of the invention is useful for the prevention and treatment of diseases which develop and/or persist by escaping immune responses triggered by T cell activation. Such diseases include, for example, all cancers, natural and induced immune deficiency states, and diseases caused by infections with a variety of pathogens. The method of the invention is illustrated herein by demonstrating its application to three different types of human cancers. Cancer cells are by nature generally weakly immunogenic, fail to trigger an effective T cell response, and survive and grow as a result. As demonstrated herein, cancers can be prevented, and established cancers may be cured, by stimulating an effective T cell response using autologous tumor cell vaccines of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods for immunizing individuals against disease and for treating individuals with established diseases using cellular vaccines created with a two-step process described herein. The methods of the invention may be applicable to any disorder involving a low- or non-immunogenic response pathology, wherein effective treatment or prophylaxis requires an immune boost through activation of T cells. Such disorders include, but are not limited to, all forms of cancer, immune deficiency disorders (both natural and induced), and infectious diseases caused by viral or other pathogenic agents.

A. Current Approaches to the Generation of Cellular Vaccines Against Tumors

Anti-tumor immune responses are primarily mediated by T cells. For optimal activation of T cells, at least two signals are essential. The first is an antigen-specific signal via the T cells receptor and MHC-polypeptide complex interaction. The second is a costimulatory signal mediated via a different set of receptor/counter-receptor pathway. Down-regulation expression of MHC and the molecules that costimulate the immune responses are associated with defective signaling of tumor cells for T cell activation. The immunogenic potential of tumor cells can be enhanced through defined molecular modifications. The modified tumor cells can be used as cellular vaccines to elicit anti-tumor specific immunity that is effective in eradicating established tumors in animals. Most of these approaches have required ex vivo or in vivo gene transfection with a viral vector or modification of tumor cells with antigen processing cells (APCs).

1). Gene Transfer to Tumor Cells

Many approaches have been used to enhance immunogenicity of tumor cells. In early studies, the stimulatory signals were provided exogenously by immunizing animals with tumor cells mixed with adjuvants or with chemically modified tumor cells. Recent advances in genetic engineering allow the modification of tumor cells by gene transfection. For example, transfection of MHC genes into tumor cells converted non-immunogenic tumor cells into immunogenic ones (Haddada et al., Proc Natl Acad Sci USA 1986, 83:9684-9688; and Wallich et al., Nature 1985, 315:301-315). Immunization of animals with tumor cells transfected with cytokine genes, including IL-1 (Z_ller et al., Intl J Cancer 1992, 50:450-457), IL-2 (Bubenik et al., Immunol Lett 1990, 23:287-292), IL-4 (Tepper et al., Cell 1989, 57:503-512), IL-6 (Porgador et al., Cancer Res 1992, 52:3679-3683), IL-7 (Hock et al., J Exp Med 1991, 174:1291-1298), TNF (Asher et al., J Immunol 1991, 146:3227-3235), G-CSF (Colombo et al., J Exp Med 1991, 173:889-897), GM-CSF (Dranoffet al., Proc Natl Acad Sci USA. 1993, 90:3539-3543), IP-10 (Luster et al., J Exp Med 1993, 178:1057-1065) and IFN.gamma. (Gansbacher et al., Cancer res 1990, 50:7820-7825) also induced host anti-tumor immune responses. In some of these studies, anti-tumor immunity was mediated by T lymphocytes. In other studies, anti-tumor immunity was mediated by macrophages or neutrophils. Inhibition of insulin like growth factor production by transfection with a vector expressing anti-sense IGF-I rendered rodent C6 glioma more immunogenic (Trojan et al., Science 1993, 259:94-96).

Tumor cells (J558L) were engineered to over-produce one of 5 different cytokines (IL-2, IL-4, IL-7, TNF, or IFN.gamma. (Hock et al., Cancer Res 1993, 53:714-716). These cytokine producing tumor cells were then used to immunize animals against a challenge with parental tumor cells. Injection of all cytokine producing tumor cells induced systemic responses capable of mediating the rejection of parental tumor cells when injected in low numbers.

Gene transfection approaches usually require the establishment of cells in tissue culture and transfection of primary tumor cells and selection in vitro. They are time-consuming and costly. The clinical application may thus be limited.

To circumvent requirements for the establishment of tumor cell lines in vitro, the direct introduction of genes into tumor cells in situ has been attempted. A gene encoding an allogeneic HLA antigen was incorporated into liposome, and injected into five patients with advanced melanoma. One patient demonstrated regression of tumor nodules. In this patient, metastatic lesions at distant sites also displayed complete regression. The other four patients did not respond to the treatment. These results suggest that introduction of exogenous genes directly into tumors has potential as a therapeutic approach (Nabel et al., Ann. NY Acad Sci 1995, 27:227-231).

2). Antigen Presentation by APCs

An effective tumor vaccine should have all the signals essential for T cell activation. Activated B cells are very effective antigen-presenting cells. In addition to expressing of high levels of MHC class II antigens, activated B cells express high levels of accessory and costimulatory molecules. In one approach, BERH-2 cells, a chemically induced rat hepatoma cell line, were fused with in vivo activated B cells. The hybrid tumor cells expressed high levels of MHC class I, II, ICAM-1 and B7 and lost tumorigenicity in syngeneic animals. Animals immunized with the hybrid cells generated by fusion of tumor cells with either activated B cells or dendritic cells became resistant to parental tumor challenge and cured established tumors (Toffaletti et al., J Immunol 1983, 130:2982-2986; and Guo et al., Science 1994, 263:518-520).

Dendritic cells transfected with tumor antigen expressing nucleic acids were able to be used as tumor cellular vaccines to stimulate T cell immunity (Liu, 1998, Nature Biotechnology 16:335). Several recent papers have showed that autologous dendritic cells pulsed ex vivo with tumor antigens including tumor associated antigen or tumor specific idiotype protein were able to be used as tumor cellular vaccines to stimulate host immunity (Young et al., J. Exp. Med. 1996, 183:7-11; Celluzzi et al., J.Exp. Med. 1996, 183:283-287; Hsu et al., Nature Med. 1996, 2(1): 52-58; and Gong et al., Nature Med. 1997, 3(5): 558-561). In contrast to gene transfection experiments in which only one or two genes can be introduced into tumor cells at a time, fusion of tumor cells with APCs or modification of dendritic cells with tumor antigens ex vivo gives rise to treated APCs with both tumor specific and costimulatory signals. However, these approaches are still time-consuming and requiring large numbers of activated B cell and dendritic cells.

B. Improvement and Alternative to the Current Approaches

The present invention provides improvements and alternatives to the current approaches to generate cellular vaccines against cancer and other diseases.

In one embodiment, the methods of the invention comprise modifying antigen-presenting cells (APCs) by (1) treating the APCs in order to present on the cell surface thereof antigens derived from or associated with the target diseased cells, and (2) attaching to the APCs a bridge molecule capable of binding to one or more T cell activation costimulatory molecules on the surface of T cells (e.g., CD28), thereby providing the APCs with the capacity to physically link to T cells and activate the costimulatory signal. For the first step, the APCs may be (a) transfected with foreign nucleic acid capable of expressing within the APCs antigens derived from or associated with the target diseased cells (e.g., Donnelly et al, 1994, J. Immunol. Methods 176:145), (b) pulsed with tumor lysate (e.g., Nestle et al., 1998, Nat. Med. 4:238), or (c) pulsed with purified tumor associated peptides or proteins (e.g., Mayordomo et al., 1995, Nat. Med. 1:1297; Porgador et al., 1995, J. Exp. Med. 182:255; Celluzzi et al., 1996, J. Exp. Med. 183:283; Paglia et al., 1996, J. Exp. Med. 183:317; Bakker et al., 1995, Cancer Res. 55:5330; Hsu et al., 1996, Nat. Med. 2:52; Reeves et al., 1996, Cancer Res. 56:5672; Zitvogel et al., 1996, J. Exp. Med. 183:87; and Boczkowski et al., 1996, J. Exp. Med. 184:465).

In another embodiment, the methods of the invention comprise modifying weakly- or non-immunogenic autologous cells of the disorder (target cells) by (1) treating the target cells in order to amplify primary and costimulatory T cell activation signals therein, and (2) attaching to the target cells a substance capable of binding to one or more antigens on the target cells and to one or more T cell activation costimulatory molecules on the surface of T cells (e.g., CD28), thereby providing the target cells with the capacity to physically link to T cells and activate the costimulatory signal. Such substances include, but are not limited to, bispecific monoclonal antibodies (Bi-MAbs) targeted to antigen on the treated cells and to CD28 and/or other costimulatory molecules on T cells.

The first step of the method amplifies the expression of cell surface molecules involved in T cell activation, such as MHC and adhesion molecules, and up-regulates antigen processing capacity within the target cells by enhancing enzyme activity involved in intracellular antigen processing. For the first step of the method, any means which can amplify primary and costimulatory T cell activation signals in the target cells (e.g., the expression of MHC and adhesion molecules), may be used. Such amplified expression may be achieved by, for example, in vitro and in vivo treatment of target cells with cytokines or other factors capable of inducing the desired amplification; and in vitro and in vivo transfer of MHC genes, adhesion molecule genes, cytokine genes, and/or MHC, adhesion molecule, and cytokine gene transcription activators or enhancers to the target cells. Specific examples include (a) introduction of gene encoding MHC Ags (Restifo et al., 1993, J. Immunother. 14:182; Ostrand-Rosenberg et al., 1990, J. Immunol. 144:4068; and Armstrong et al., 1997, Proc. Natl. Acad. Sci. USA 94:6886), costimulatory molecule (Chen et al., 1992, Cell 71:1093; Townsend et al., 1993, Science 259:368; Baskar et al., 1993, Proc. Natl. Acad. Sci. USA 90:5687; and Johnston et al., 1996, J. Exp. Med. 183:791), or cytokines (Fearon et al., 1990, Cell 60:397; Golumbek et al., 1991, Science 254:713; and Dranoff et al., 1993, Proc. Natl. Acad. Sci. USA 90:3539) into tumor cells; (b) fusion of tumor cells with antigen presenting cells (APCs) (Guo et al., 1994, Science 263:518; WO 95/16775; Gong et al., 1997, Nat. Med. 3:558; and Wang et al., 1998, J. Immunol. 161:5516); and (c) stimulation with virus infection (Von Hoegen, et al., 1990, Cell. Immunol. 126:80; Ockert et al., 1996, Clin. Cancer Res. 2:21; Ertel et al., 1993, Eur. J. Immunol. 23:2592; and Jurianz et al., 1995, Int. J. Oncol. 7:539).

In one embodiment of the method, amplification of primary and costimulatory T cell activation signals in the target cells is achieved using cytokine treatment. Target cells may be treated with cytokines ex vivo or in vitro as described in the examples herein. Alternatively, cytokines may be administered to the target cells in vivo by, for example, intralesional injection, intralymph injection, intravesical injection, subcutaneous injection, etc., in suitable pharmaceutical carriers or controlled release preparations. Any cytokine or combination of cytokines which results in the amplified expression of MHC and adhesion molecules may be used to treat cells in the first step of the method. In preferred embodiments, described more fully by way of the examples herein, a combination of interferon (IFN-.gamma.) and tumor necrosis factor-.alpha. (TNF.alpha.) is used in the first step. Preferably, cells may be treated with concentrations of between about 10-100 U IFN-.gamma. in combination with concentrations of between about 10-100 U TNF.alpha., more preferably with 100 U IFN.gamma., and 50 U TNF.alpha., as described in Example 1. However, the conditions and specific cytokines most optimal for the amplification of activation signals on the particular cells to be treated may vary and may be determined essentially as described in Example 1.

The second step of the method of the invention provides the treated cells with the capacity to physically bridge to T cell surfaces via CD28 and/or other T cell costimulatory molecules, thereby providing optimal conditions for stimulating T cell activation. For the second step, any substance capable of binding to one or more antigens on the treated cells and to one or more T cell activation costimulatory molecules on the surface of T cells may be used. Such bispecific or multispecific bridging substances may comprise, for example, Bi-MAbs, proteins and other macromolecules, and polymer materials, which contain a functionality capable of binding to the targeted T cell costimulatory molecule and activating, or inducing the activation of, the costimulatory signal. In one embodiment, described by way of the examples in Example 6, Bi-MAbs are used as the bridging substance.

One functionality of the bispecific or multispecific bridging substance may be directed to a target cell-specific antigen or any antigen expressed on the target cells. Optimally, where the target cells are to be armed with bridging substance in vivo, the target cell antigen to which the bridging substance is directed should be unique.

However, the target cell antigen need not be unique to the treated cells, since the attachment of the bridging substance may be practiced in vitro. Accordingly, bridging substances attached to the target cells in vitro will not cross-react with the same antigen on cells in the individual to be immunized with the modified cells.

After such bridging substances are incubated with cells treated according to the first step of the method, free bridging substance may be washed away and bound bridging substance may be cross-linked to the cell surface with polyethylene glycol (PEG) or another cross-linking agent.

Another functionality of the bispecific or multispecific bridging substance is specifically directed to a T cell activation costimulator such as CD28. Thus, when the modified cells are used to immunize an individual, the CD28 (or other costimulatory molecule) binding sites of the attached bridging substance are free, and will bind to CD28 (or other costimulatory molecule) on T cell surfaces, ensuring that the modified cells will become physically linked to T cells. This bridging substance-mediated physical link also brings other molecules on the surfaces of the modified cells, some of which have been amplified by the cytokine treatment step, into contact with other molecules on the surfaces of T cells, providing further costimulation which thereby further facilitates T cell activation.

The second step of the method may be practiced in vitro or in vivo, depending upon whether target cells were treated according to the first step of the method in vivo or in vitro, the circumstances of the disease or lesion to be treated, and the clinical objectives of the treatment. Where the first step of the method is conducted in vivo, the treated cells may be armed with the bridging substance in vivo as well. In this case, the clinician may use a variety of known methods for administering the bridging substance to a patient. The best route of administering the bridging substance to patients who have had disease- or lesion-specific cells (target cells) treated in vivo according to the first step of the method will depend on clinical and/or other aspects of the disease or lesion to be treated as well as on the site of the treated cells. For example, where target cells located in lymph node have been treated in vivo, direct administration of the bridging substance to the lymph node is preferred. Similarly, for example, where tumor cells have been treated in vivo by intratumor injection of cytokines or gene transfer vectors, the bridging substance preferably should be administered directly into the tumor or to the local environment of the tumor.

Where the first step of the method is conducted in vitro, the treated cells may be armed with the bridging substance in vivo or in vitro. Where the bridging substance is administered in vivo, the same route used to administer the treated cells or a similar route should be used, taking into account the same factors discussed above regarding in vivo arming of in vivo treated target cells.

When in vitro treated cells are armed in vitro, the treated and armed cells (cellular vaccine) may be used in vivo for treatment and prevention of disease, or in vitro for generation of lesion- or disease-specific cytotoxic T lymphocytes (CTLs). Arming treated cells in vitro provides the advantage of being able to use a bridging substance directed to any antigen on the target cell.

When used for treatment or immunization of a patient, in vitro treated and armed cells may be administered to the patient using a variety of methods known to those skilled in the art. In a particular embodiment, described more fully in the examples which follow, in vitro treated and armed cells are administered subcutaneously. In another embodiment, the treated and armed cells are administered by direct intralesion injection, an administration route that may provide advantages over subcutaneous administration in certain circumstances (for example, where the lesion to be treated is not well vascularized, is inaccessible for biopsy, or cannot be disrupted without creating further risk to the patient). In a further embodiment, the treated and armed cells are administered by injection into the lymph nodes. This method of administration requires fewer treated and armed cells than may typically be required using other routes of administration. Such intralymph administration may be preferred in situations where only limited autologous tissue can be obtained from a lesion using thin needle biopsy techniques (e.g., inaccessible/inoperable cancers). Moreover, intralymph administration is likely to enhance the interaction between the cellular vaccine and T cells given the large number of T cells within lymph nodes. Applicant's initial experimental data indicates that a single intralymph injection of as few as 1x10⁴ cellular cancer vaccine cells, prepared using the method of the invention, can induce an effective immune response against parenteral tumor cell challenge and can cure established tumors. The therapeutic efficacy of this method of administration appears equivalent to that achieved using 100-times more cells administered subcutaneously.

In a specific embodiment of the method of the invention, Bi-MAbs that react with CD28 are used as a bispecific bridging substance. B7 interacts with both CD28 and CTLA-4 on T cells. Under certain circumstances, the B7-CTLA-4 interaction generates a negative signal which prevents T cell activation. Thus, by immunizing with cells coated with Bi-MAbs specific for CD28, the interactions between B7 on such cells and CTLA-4 (or other T cell activation down-regulating molecules) is minimized and/or bypassed. In addition, Bi-MAbs reactive with other costimulatory T cell surface molecules (e.g., CD2, CD48) may also be used in the practice of the method of the invention. Furthermore, more powerful costimulation may be achieved by using a multiplicity of Bi-MAbs having specificity for various T cell costimulators.

Bridging substances may be prepared using well known technologies. As shown in the examples which follow, Bi-MAbs may be used effectively as the bridging substance. Such Bi-MAbs may be generated using methods well known in the art, such as, for example, those described in Example 2, and as described in the references cited therein. Bi-MAbs containing multiple T cell costimulatory molecule binding sites may be prepared by chemical linkage in order to provide a means for generating multiple costimulatory activation circuits.

In addition to Bi-MAbs, molecules engineered to contain functional binding sites specific for both the antigen(s) of the target cell(s) and the T cell costimulatory molecule(s) may be used as the bridging substance in the practice of the method of the invention. Such molecules may, for example, comprise proteins, other macromolecules, and polymers engineered to contain the desired binding sites, and may be prepared by using genetic engineering technologies, synthetic technologies, or by chemical linkage of component polypeptides, polymers, and/or other macromolecules. The binding site components of such bridging substances may comprise, for example, Fab2 antibody fragments, antibody binding sites, natural or engineered ligands, or other factors reactive with the target cell antigen(s) and T cell costimulatory molecule(s).

The bridging substance may be administered to a patient in vivo using a pharmaceutically acceptable carrier or a variety of drug delivery systems well known in the art. As an example, for cancer immunotherapy, a combination of the cytokines TNF.alpha. and IFN.gamma. and an anti-CD28 Bi-MAb may be formulated within a controlled release preparation which is administered to the patient by directly injecting the preparation into the lymph nodes or into the tumor itself.

The method of the invention is particularly useful in treating cancers. This aspect of the invention is more fully described by way of the examples presented in Example 6. The data presented in the examples indicate that strongly immunogenic tumor cells can be generated using the two-step method of the invention, comprising (1) in vitro treatment of autologous tumor cells with a combination of .gamma.-interferon (IFN.gamma.) and tumor necrosis factor-.alpha. (TNF.alpha.), and (2) pre-incubation with a Bi-MAb specific for both an antigen on tumor cells and CD28 on T cells. The resulting modified tumor cells are able to act as a cellular vaccine that elicits CTL-mediated immunity which can both prevent and cure established tumors. Autologous cancer cells and other diseased cells can be removed from patients by surgery or other techniques, such as fine needle biopsy, including, but not limited to, devices and methods described in U.S. Pat. Nos. 5,669,394, 5,655,541, 5,241,969, 5,060,658, 4,989,614, 4,697,600, and 4,605,011, incorporated by reference herein.

In particular, the studies described in the examples which follow show that cytokine-treated, anti-CD28 Bi-MAb-armed hepatoma cells induce protective immunity against parental tumor cell challenge and, moreover, cure established gross hepatomas in mice. In addition, the studies described in Example 7, show that the method of the invention also induces protective immunity against lymphoma and colon carcinoma.

Different routes of administration, or combinations thereof, may be preferred when treating different cancers or other diseases using the cellular vaccines of the invention. As illustrated by the study briefly described in Example 4, immunization with cytokine-treated (and un-armed) cells followed by intravenous administration of an anti-CD28 Bi-MAb induces some anti-tumor immunity in the hepatoma model system.

In comparison, as shown by the results of the studies described in Examples 5 and 6, the administration of cells treated with cytokines in vitro and armed with Bi-MAbs in vitro induces uniform tumor immunity and cures established hepatomas. It is possible that insufficient localization of the Bi-MAbs to the tumor tissue following intravenous injection in the former case is responsible for the difference in therapeutic efficacy.

Individuals may be immunized against a variety of diseases with cytokine-treated, anti-CD28 Bi-MAb-armed autologous cells, thereby providing the individual's immune system with a signal sufficient to activate T cells and confer protective immunity. Similarly, individuals may be treated for a variety of diseases by administering cytokine-treated, anti-CD28 Bi-MAb-armed autologous cells of the disease or lesion, thereby providing the individual's immune system with a signal sufficient to activate T cells and induce a cytotoxic T lymphocyte response. In both cases, Bi-MAbs with a specificity for other T cell costimulatory molecules may be used to arm the treated cells with a means to physically bridge to T cells in vivo.

In addition to MHC class I, ICAM-1, ICAM-2 and VCAM-1 molecules, treatment with cytokines may enhance tumor antigen processing by tumor cells, and may induce the expression of other cell surface molecules essential for T cell activation. A combination of cytokine treated hepa 1-6 cells and immobilized anti-CD28 MAb failed to stimulate splenic T cells in vitro or to induce anti-tumor immunity in vivo in applicant's model system. This suggests that the signal delivered by the interaction between CD28 and anti-CD28 MAb is not sufficient in itself to induce T cell activation.

In contrast, a strongly immunogenic response is obtained when cytokine treated tumor cells armed with anti-CD28 Bi-MAb in vitro interact with CD28 on T cells, indicating that a physical bridging function is an important component of the activation process.

In addition, the observation that cytokine treated, B7 transfected hepa 1-6 cells were not able to activate splenic T cells in vitro (Example 2) is consistent with the recent finding that B7 may interact with CTLA-4 to deliver a negative regulatory signal, and provides a strong rationale for using anti-CD28 Bi-MAbs to physically link the antigen presenting cell specifically to CD28 molecules for T cell activation.

The invention provides an effective alternative to gene transfer and tumor:APC engineering for the development of cellular vaccines. In particular embodiments of the invention, described in the following examples, the attachment of Bi-MAbs to tumor or other target cells takes place in vitro. Accordingly, the antigens on such cells need not be unique to those cells. Essentially, any antigen may be targeted. Bi-MAbs can be produced by linking anti-CD28 MAbs to Mabs that recognize any antigen expressed on the tumor or other target cell, including antigens which are also expressed on large populations of cells in the individual to be treated (e.g., lymphocytes). This approach may be particularly useful in situations where Mabs to tumor specific antigens are not available.

Claim 1 of 8 Claims

What is claimed is:

1. A method of preparing an immunogenic composition, comprising the steps of:

(a) providing an autologous target tumor or pathogen infected cell;

(b) treating the tumor or pathogen infected cell with IFN-.gamma. and/or TNF-.alpha. to increase concentration of a primary T cell activation molecule or a costimulatory T cell activation molecule in the target tumor or pathogen infected cell;

(c) providing a bispecific monoclonal antibody including one or more binding sites for one or more costimulatory molecules on a surface of one or more T cells of a patient mammal, wherein the bispecific antibody is a CD28:gp55 bispecific monoclonal antibody;

(d) attaching the bispecific monoclonal antibody to the target tumor or pathogen infected cell; and

(e) collecting a pharmaceutically effective amount of the target tumor or pathogen infected cell with the attached bispecific monoclonal antibody.

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