WO1997024450A1

WO1997024450A1 - Method for immunizing and treating human cells in animal organs maintained in vitro

Info

Publication number: WO1997024450A1
Application number: PCT/US1996/019967
Authority: WO
Inventors: Kye-Hyung Paik
Original assignee: Paik Kye Hyung
Priority date: 1996-01-02
Filing date: 1996-12-18
Publication date: 1997-07-10
Also published as: AU1420497A

Abstract

A method of immunizing cells in vitro in which immune cells of one species are cultured in tissue or an organ of another species during sensitization with an immunogen is disclosed. Methods of immunizing human immune system cells in vitro for use in eliciting an appropriate immune response when the cells are returned to the human or for making hybridomas that produce human monoclonal antibodies are disclosed. An animal model for human Hepatitis B virus infection in rats is disclosed.

Description

METHOD FDR IMMUNIZING AND TREATING HUMAN CELLS IN ANIMAL ORGANS MAINTAINED IN VITRO Field of the Invention The present invention relates to methods of organ culture in vitro, and specifically relates to methods of treating cells in culture to immunize cells in vitro for use in making hybridomas of human origin or for transplantation into humans with pathologic immune conditions and development of an animal model for chronic viral disease for testing therapeutics developed using the in vitro cell treatment method.

Description of the Prior Art

The use of monoclonal antibodies directed against pathogenic cells, especially tumors, was initially believed to be a breakthrough in therapeutics, especially for cancer treatment (Trowbridge and Domingo, Nature, Lond.

294:171-173, 1981; Ritz and Schlossman, Blood 59:1-11, 1982; Bumol et al., Proc. Natl. Acad. Sci. USA 80:529-

533, 1983). However, human immune responses to monoclonal antibodies of animal origin and technical difficulties encountered in production of monoclonal antibodies of human origin have limited the use of monoclonal antibodies as therapeutics {Shawler et al., J. Immun. 135:1530-1535, 1985; Meeker et al., New Engl. J. Med. 312:1658-1665, 1985). Thus, although production of monoclonal antibodies it has been one of the greatest discoveries in the field of biomedical research, it has not been effectively used to produce life-saving therapeutics for use in humans.

The method of producing monoclonal antibodies can be divided into three steps: (1) immunization; (2) cell fusion; and (3) antibody production (Kohler and Milstein, Nature, Lond. 265:495497, 1975; Oisson and Kaplan, Proc. Natl. Acad. Sci. U.S.A. 77:5429, 1980). One limitation to the production of human monoclonal antibodies is that in vivo immunization of humans with some antigens (e.g., tumor antigens, human antigens or infectious agents) may have undesirable consequences. These include human immune reactions to animal proteins, which are sometimes fatal or result in ineffective antibodies. In vitro stimulation of cultured cadaveric spleen cells and toπsilar tissue from toπsillectomies has been successfully used in the production of human hybridomas (Wasserman et al., J. Immun. Methods 93:275-283, 1986). This disclosure describes improved methods of immunization of cells maintained in vitro.

Sensitizing immunocytes outside of a living body has been generally ineffective using conventional methods and immunization of ceils inside a living animal has primarily been used in the production of hybridomas. However, because of human immunological reactions to monoclonal antibodies of animal origin (e.g., human immune reactions to murine antibodies), immunization of animals to produce monoclonal antibodies has had limited application for producing therapeutic antibodies for humans. Immunological reactions against cross-species antibodies are sometimes fatal to the patient even at the first usage and almost always render the monoclonal antibodies ineffective for extended periods of treatment (Shawler et al., J. Immun. 135:1530-1535, 1985).

Naturally occurring human cells that produced antibodies or chimeric antibodies that are partially of human origin have been used as an alternative source of monoclonal antibodies for human treatment. One human-origin monoclonal antibody that has been used therapeutically is a human anti-hepatitis B envelope antibody (Ostberg, L. and Pursch, E. Hybridoma 2:361-367, 1983). This antibody, initially found in human plasma, was used to treat patients experiencing recurrent hepatitis after liver transplantation but was found to be ineffective. The cell line used to produce this human anti-hepatitis B envelope antibody was found naturally occurring in a human and therefore immunization per se was not required.

There has only been limited success in producing human B cell hybridomas with a few antigens (Olsson and Kaplan, Proc. Natl. Acad. Sci. U.S.A. 77:5429, 1980; Chiorrazzi et a!., J. Exp. Med. 156:930, 1982; Kozbor et al.,

Proc. Natl. Acad. Sci. U.S.A. 79:6651 , 1982). Therefore, chimeric antibodies that are "humanized" have been used as an alternative source of monoclonal antibodies for human treatment. Such chimeric antibodies generally comprise a molecule that is partly of human origin (usually the constant or Fc portion) and partly of animal origin (usually the antibody-recognition or Fab portion) (Neuberger et al.. Nature, Lond. 314:268-270, 1985). However, even humanized chimeric antibodies can result in adverse immune responses, especially to the cross-species portions of the molecules.

Monoclonal antibodies of completely human origin can potentially limit adverse immune responses in a patient because they contain no cross-species protein. Human monoclonal antibodies may be particularly useful for treating cancers and preventing or treating rejection after organ transplantation.

A variety of culture methods are well known in the art. An automated organ culture method which includes a rotational system for periodically immersing a slice of organ tissue contained within a stainless steel mesh chamber in culture medium has been described previously (S.S. Park, Inje Med. J. 14(3): 363-369, 1993). An in vitro thymic organ culture system for identifying potential anti-viral agents has been disclosed (PCT patent application WO

9505453).

The present invention is for methods of maintaining cells in vitro where they can be immunized to activate antibody-producing cells that can be subsequently fused with other cells for the production of monoclonal antibodies. In the method, a chimeric organ is produced in which cells from one species are maintained in an organ from another species and the chimeric organ is maintained in vitro. The disclosed organ culture methods can also be used to maintain and treat cells or tissues in vitro prior to transplantation into a recipient mammal. Similarly, the organ culture method can be used as a model system for investigating treatment of infected tissue, e.g. for drug discovery. Summary of the Invention

According to one aspect of the invention, there is provided a method of maintaining cells in culture. The method includes providing organ tissue from a first animal to serve as a host tissue in in vitro culture, providing target cells from a second animal, incubating the host tissue with the target cells to produce a chimeric organ, and maintaining the chimeric organ in in vitro culture conditions. In one embodiment, the target cells are immune system cells, preferably lymphocytes, dendritic cells or a mixture thereof. In another embodiment, the method further includes irradiating the host tissue with a sublethal dose of irradiation to kill resident lymphocytes before the target immune system cells are incubated with the host tissue. In yet another embodiment, the method includes irradiating the host tissue with a supralethal dose of irradiation to kill resident immune ceils before the target immune system ceils are incubated with the host tissue. In a preferred embodiment, the host tissue is spleen tissue. In other preferred embodiments, the first animal is selected from the group consisting of rats, mice, dogs, chickens, frogs and humans, and the second animal is selected from the group consisting of rats, mice, dogs, chickens, frogs and humans. The method can further include the steps of treating in vitro target cells from a human having a pathologic condition and returning the target cells to a human having a pathologic condition after treatment of the target cells in vitro where the treated cells can elicit or produce an appropriate immune response. In another embodiment, the method can further include immunizing the target immune system cells by exposing the chimeric organ to an immunogen in in vitro culture, in a preferred embodiment, the immunogen is an antigen selected from the group consisting of human cell antigens, tumor antigens and viral antigens, preferably hepatitis B virus antigens. Preferably, the method can further include a step of collecting in vitro treated target cells from the chimeric organ for transplantation of the target cells into a recipient mammal. The method can also include another step of testing the in vitro treated target immune system cells in an animal model of a human disease before using the cells or their products in humans. According to another aspect of the invention there is provided a method of immunizing cells in culture. This aspect of the invention includes providing organ tissue from a first animal to serve as a host tissue in in vitro culture, providing target immune system cells from a second animal, incubating the host tissue with the target immune system cells to produce a chimeric organ, and immunizing the target cells in the chimeric organ by exposing the chimeric organ to an immunogen in in vitro culture conditions. The method can further include irradiating the host tissue with a sublethal dose of irradiation to kill resident lymphocytes before the target immune system cells are incubated with the host tissue. In another embodiment, the method can include irradiating the host tissue with a supralethal dose of irradiation to kill resident immune cells before the target immune system cells are incubated with the host tissue. Preferably, the immunogen is an antigen selected from the group consisting of human cell antigens, tumor antigens and viral antigens, preferably a hepatitis B virus antigen. This aspect of the invention can also include removing the target immune system cells from the chimeric organ after immunizing the target cells and fusing the target cells with cells capable of producing a hybridoma for production of monoclonal antibodies and, furthermore, producing the monoclonal antibodies from the hybridoma. Preferably, the first animal is selected from the group consisting of rats, mice, dogs, chickens, frogs and humans and the second animal is a human.

According to another aspect of the invention there is provided monoclonal antibodies for treating a pathological condition in humans produced by the method that includes the steps of providing organ tissue from an animal to serve as a host tissue in vitro, providing target immune system cells from a human, incubating the animal host tissue with the human target cells to produce a chimeric organ, immunizing the human target cells in the chimeric organ by exposing the chimeric organ to an immunogen in in vitro culture conditions, removing the human target cells from the chimeric organ after immunizing the human target cells, fusing the human target cells with cells capable of producing a hybridoma for production of monoclonal antibodies, and producing and isolating human monoclonal antibodies from the hybridoma. Preferably, the monoclonal antibodies are human monoclonal antibodies that specifically bind to an immunogen that is a human cell antigen, tumor antigen or viral antigen including hepatitis B virus antigens.

According to another aspect of the invention there is provided a mammal infected with a human virus produced by the steps of treating a mammal with an immunosuppressant drug, partial hepatecto y, splenectomy or any combination thereof; infecting the treated mammal with a human virus within 24 hours after treatment with an immunosuppressant drug, partial hepatectomy, splenectomy or any combination thereof; and maintaining the infected mammal alive for one to eleven days after infection. Preferably, the mammal is infected with a human hepatitis virus. In one embodiment the mammal is a transgenic mouse that is tolerable to the human virus. In another embodiment, the mammal is a fetal mammal. It should be understood that both the foregoing general description and the following detailed description, along with the drawing, constitute the disclosure and illustrate various embodiments of the invention.

Brief Description of the Drawing FIG. 1 shows a device for maintaining tissue samples alive in vitro in an automated culture system.

Detailed Description of the Invention Organ culture means placing and maintaining an organ or a portion of an organ in a medium to supply nutrients to the cells of the organ, at a temperature near or at a physiological temperature, in an atmosphere near or at physiological conditions. Generally this means placing a slice of organ tissue in a tissue culture medium at about 37"C in an incubator containing about 5% to 10% C0₂. Dynamic organ culture systems have been disclosed in which liver tissue viability is maintained for about 24-48 hours (Smith, P.F. et al., Life Sci. 36: 1367, 1985; S.S. Park, Inje Med. J. 14(3): 363-369, 1993).

Unless defined otherwise, all scientific and technical terms used herein have the same meaning as commonly understood by those skilled in the relevant art. Unless mentioned otherwise, the techniques employed or contemplated herein are standard methodologies well known to one of ordinary skill in the art.

In vitro immunization in a chimeric organ involves the following steps. (1) Taking of the immune organ or tissues from an animal using whole body perfusion with commercial preservation solution. For example, rodent (rat or mouse) splenectomy is anticipated. (2) Maintaining the organ in culture using an automated system or other well- known methods of tissue culture and co-culturing target immune system cells with the host organ tissue to produce a chimeric organ. The host immune organ can be irradiated with a sublethal dose of irradiation to kill the resident lymphocytes before the target immune system cells are introduced. Alternatively, irradiation of the organ can be omitted from the procedure. Preferably, the target cells are peripheral blood lymphocytes (PBL) from the species for which monoclonal antibodies are to be produced. For example, human PBL can be isolated by centrifugation from human blood using methods well known in the art and then injected into an irradiated rodent or into a rodent spleen maintained in vitro. Alternatively, the next step can be dipping the tissue in lymphocytes isolated from centrifuged blood and incubating the tissue and cells together for about 10 min in cell culture medium at 37°C in an incubator containing 5-10% CO-. (3) Immunizing the target immune system cells in vitro by exposing the chimeric organ to an immunogen introduced into the organ culture system at concentrations well known to those skilled in the art or easily determined by routine testing. The method can be used similarly for in vitro immunization of target lymphocytes and dendritic cells in a chimeric organ.

Preferably, organ culture is performed by periodically submerging the chimeric organ in modified Waymouth's MD 752/1 media, at 37°C, in an appropriate gas mixture (e.g. at 2.5 atm of pressure of 95% D₂ and 5% C0₂) over a period of about 24 hours. Referring to FIG. 1, an excised organ or portion of organ tissue 10 is placed inside of a porous container 11 that is placed inside of a culture tube 12 which is rotatable and has at least one inlet port 13 for entry of gases, medium, immunogen and the like. The porous container 11 may be made of any inert substance including but not limited to stainless steel mesh, plastic mesh, nylon mesh or a semi-permeable membrane. Preferably, the porous container 11 is a stainless steel mesh box having an average pore size of about 100-300 μm, preferably about 200 μm. A square or rectangular box, rather than a spheroid container, is preferred because the shape will rotate only when the culture tube 12 is rotated and will remain oriented within the culture tube 12 rather than randomly oriented as a spherical containing would be when the culture tube is rotated. The culture tube 12 includes a resealable sampling port 14 for removal of samples of tissue culture medium 15. The sampling port can also be used for injection of medium and/or immunogen. The organ tissue 10 is periodically immersed in the tissue culture medium 15 as the culture tube 12 is rotated. Gas exchange within the culture tube 12 occurs at intervals in which a gas mixture is introduced into the inlet port 13. To maintain constant gas pressure during gas input, gas may be expelled via an outlet port 16 of the culture tube 12. The culture tube 12 is maintained in an incubator (not shown) having a constant temperature of 37°C. The organ culture processes including rotating the culture tube, exchanging gases within the culture tube, introducing medium and/or immunogen, and sampling medium can be automated.

Those of skill in the art of immunology will be able to empirically determine the most effective culture time for immunization using routine testing methods. Generally, after less than 24 hours of incubation newly produced antibodies can be found in the culture medium in which the cells have been incubated as determined by protein precipitation assays. After less than 24 hours of incubation, immunologically sensitized human lymphocytes in the chimeric tissue can generally be detected by microscopically observing morphological changes associated with immunological activation as is well known in the art.

One embodiment of the method includes irradiation of the recipient organ with a supralethal dose followed by injecting or incubating the organ with bone marrow ceils of the target organism which are purified using a Fluorescence Activated Cell Sorter (FACS). The chimeric organ is then cultured in vitro using an automated organ culture system as described herein or equivalent known tissue culture methods and the target cells are exposed to an immunogen in culture.

The invention provides a method of sensitizing immune cells of a target organism outside of the living body, which is a key step to make human origin monoclonal antibodies that specifically recognize an immunogen. One embodiment of the method is termed "in vitro immunization" (IVI) to produce human antibody-secreting cells in vitro that can be used in production of hybridomas to make human monoclonal antibodies.

In another embodiment of the method, called "in vitro cell treatment" (IVCT), cells are treated in vitro before being transplanted into a recipient patient. Immune system cells are preferred target cells for IVCT. Similar to methods used in bone marrow transplantation, IVCT ceils can be used to treat patients with selective or diffuse hypo- immune conditions due to chronic viral infection, organ transplantation and immunosuppression, genetic or acquired immune diseases or autoimmune conditions. The patient's immune cells are placed in an animal organ maintained in vitro to produce a chimeric organ. The chimeric organ is then exposed to an immunogen such as a viral antigen and, when an appropriate immune response is detected, the IVCT cells are placed into the recipient patient to provide an appropriate immune response.

Any immunogen well known in the art, or which can be identified using well known assays and techniques, is useful as an immunogen in the disclosed organ culture and IVCT methods. For example, many human tumor antigens are already well known or can be identified using routine testing methods and are envisioned as antigens in the present invention (Ezzell, C, 1 NIH Res. 7:46-49, 1995; Schwartz, R.H., Cell 71:1065-1068, 1992; Boon, T., Adv. Cancer Res. 58:179-210, 1992; van der Bruggen, et al. Science 254:1643-1647, 1991; and references cited within these references). Also suitable as immunogens in the disclosed methods are human cell antigens and viral antigens that are well known or can be routinely identified and isolated using well known techniques (Handbook of Experimental Immunology. Vol. 14, D.M. Weir et al., eds., 4th ed., Blackwell Scientific Publications, 1986; Molecular Bioloov of the Cell. B. Alberts et al., eds., 3rd ed., Garland Publishing, Inc., 1994; Fundamental Virology. B.N. Fields et al., eds., 2nd ed., Raven Press, Ltd., 1991; Virology, A Laboratory Manual, F.G. Burleson et al., Academic Press, Inc., 1992). Already identified antigens suitable as immunogens include the human tumor antigens CEA, CA19-9 and σ-fetal protein; human cell antigen CD3; and viral surface antigens such as found in hepatitis virus B and C chronic infections.

Another embodiment of the present invention is useful for maintaining in vitro chimeric organs comprising human cells in animal tissues (xenochimeric organs). For example, human lymphocytes could be maintained in vitro in organ tissue from a chimpanzee or baboon. This method can be used for culturing human cells to obviate the need to obtain fresh human cells or tissue for immunization in vitro using the methods described herein. In addition, many combinations of tissues and ceils are possible including cells from one animal in organ tissue from another animal (xeno- or allochimeric organ), and human cells in organ tissue from another human (allochimeric organ). An example of a xenochimeric organ is a animal tissue containing dendritic cells (e.g., a baboon spleen) combined with human lymphocytes to maintain and treat the human lymphocytes for use in cell fusion to produce a hybridoma. A human allochimeric organ is particularly useful for producing a human alloantibody, such as an anti-CD3 antibody similar to the well known human 0KT3 antibody.

The methods of the present invention are useful for immunizing cells in vitro, especially human immune system cells, to produce an immune reaction or for treating cells in vitro before transplantation into a patient. The methods of the present invention are useful for treatment of patients who need but cannot produce an appropriate immune response in vivo but can respond when cells are taken from the patient, treated in vitro and returned to the patient. The method is valuable for immunizing human immune cells in vitro to produce antibodies that specifically recognize predetermined antigens. The antibody-producing cells can then be fused with immortal cells to produce hybridomas of human origin that, in turn, can be used to produce human monoclonal antibodies for use as therapeutics. There is a need for human origin monoclonal antibodies in many therapeutic situations including cancer therapy and organ transplantation. For example, human origin monoclonal antibodies can be used in immunotherapy after organ transplantation. Human monoclonal antibodies produced by this method can be especially important in treating chronic conditions which require repeated use of therapeutic antibodies. Thus, the present invention is useful for discovery of new therapeutics to prevent and improve the current treatments of human pathological conditions.

The methods of the present invention are also useful for producing an animal model system for discovery of new therapeutics especially for viral infections. Contrary to generally accepted concepts of viral infection, some species of virus do not exhibit an absolute species-specificity for infection (Paik et al., "Human Hepatitis B Virus

Infection to Non-human Livers", Abstract, Int'l Liver Transplantation Soc. Mtg., 1995). Preferably, the human virus is one that replicates in liver tissue (e.g., hepatitis B virus) because the liver is thought to protect viruses from the immune system. Furthermore, the viral infection can be continued chronically by infecting transgenic mice which are tolerable to the virus with a large dose of virus (i.e., modeling human chronic infections) and by using immunosuppressant therapeutics, partial hepatectomy, splenectomy or fetal inoculation in normal mice. Such an infected animal can be used as a model for chronic viral disease in which IVCT cells or antibodies produced from them can be tested in an animal before used in human treatment. An animal infected with a human virus is also useful for preparation of vaccine material or for drug discovery in physiological conditions mimicking human infection.

The invention can be better understood by way of the following examples which are representative of the preferred embodiments, but which are not to be construed as limiting the scope of the invention.

Example 1 In Vitro Immunization of a Chimeric Organ with Human Hepatitis B Virus Antigens A white rat was generally anesthetized with ether and surgically opened in the belly region using methods well known in the art. Then, 10 ml of chilled (about 4°C) Wisconsin solution (Viaspan, DuPont) was injected into the aorta after cutting the caval vein to allow perfusion. The spleen was removed from the bloodless field and stored in chilled Wisconsin solution (about 4°C). The spleen was irradiated with 300 rad from an X-ray source using methods well known in the art of immunology. The spleen was sliced into portions (e.g., 2 cm² pieces of about 260 μm thickness) in the chilled culture media. The pieces were incubated in a suspension of human PBL, isolated by centrifugation using standard methods, at 37°C for about 10 to 15 minutes in an incubator containing 5-10% C0₂. Then, the chimeric organ was cultured in the automated organ culture system as shown in FIG. 1 with 10 μg total of human hepatitis envelope antigen as the immunogen. Alternatively, an extract of a biopsy tissue of a hepatitis patient may be used as immunogen using methods well known in the art.

The culture conditions were: 37°C, in culture medium at pH 7.0, under 1.6 to 2 atm of a gas mixture of 5% CO_j and 95% 0₂. The culture medium was Modified Waymouth's MB 75211 media prepared from: Waymouth MB 75211 powdered medium (Gibco), 10% fetal bovine serum, 2.2% sodium bicarbonate, 25 mM D-glucose, 1 μg/ml crystalline bovine zinc insulin, and a standard antibiotics mixture containing 50 U/ml penicillin and 50 μg/ml streptomycin (Gibco) in distilled water. Gas exchange was made at intervals of 2.5 minutes and tissues were immersed into culture medium 4.5 times per minute by rotating the culture tube. incubation with immunogen was generally from about 1 to 24 hours, preferably about 12 hours. Generally, after less than 24 hours of culture, new protein synthesis by the cells in the chimeric organ was detected as protein in the culture medium that was precipitable with anti-hepatitis B virus antibody. Also, immunologically sensitized human lymphocytes in the chimeric tissue were observed microscopically as determined by morphological changes associated with immunological changes occurring after less than 24 hours of incubation. For other immunogens, those skilled in the art will be able to empirically determine the most effective culture time for immunization with a particular immunogen using routine testing methods (e.g., protein precipitation). Example 2

Human Hepatitis B Virus Infection of Non-human Liver Tissue The disclosed culture method was also used to demonstrate human hepatitis B virus infection into non- human liver tissue. For the preparation of human hepatitis B virus containing media, liver biopsy tissue of patients with hepatitis B surface antigenemia was incubated for 3 hours in modified Waymouth's MB 752/1 media at 37°C in an incubator containing about 5% C0₂ and 95% 0₂. Then slices of livers of three dogs, five rats, three mice, four chickens and two frogs were cultured in this media using an automated organ culture system as shown in FIG. 1. In separate culture tubes, the culture medium without virus used to maintain animal liver tissues under the same culture conditions as a control. The titers of hepatitis B surface antigen in culture media were measured after 6, 12, and 24 hours of culture using an enzyme linked immunosorbent assay using techniques well known in the art (e.g., an ELISA kit available from Abbott). The presence of viral DNA was determined by dot-blot analysis in all cultured tissues after 24 hr of incubation. The presence of viral antigens and Dane particles was determined using well known immunohistoche ical and immunoelectron microscopic methods. The titers of hepatitis B surface antigen increased rapidly to about a two-fold total increase after 6-hours of incubation in the culture media containing hepatitis B virus. Spots of viral DNA were present and core and surface antigens were stained intensely on cytoplasm and nuclei for all the animal tissues used. Using standard methods of immunoelectron microscopy, Dane particles and antigens were tagged with gold particles in culture medium for all of the animal tissues tested. In contrast, the control media did not show any change in the titer.

These results suggest that hepatitis B viral infection is not species specific. This system of virus culture in vitro can be used as a model to understand the pathogenicity of hepatitis B virus and develop new therapeutic treatments against hepatitis B virus. Furthermore, these results suggest that xenotransplantation may not be a valid option in patients with hepatitis B antigenemia. Therefore, the need to be able to maintain and expand human donor tissue for transplantation using the organ culture methods disclosed herein is further emphasized.

Example 3 Immunizing Human Immune Cells In Vitro with Hepatitis B Virus Antigens In this method, the human hepatitis B virus is infected into a nonhuman liver essentially as described in

Example 2. Human immune system cells, including human PBL, are incubated with the hepatitis B virus-infected nonhuman liver to form a chimeric organ essentially as described in Example 1. Then the chimeric organ is maintained in culture in vitro for 2-24 hr to immunize the human cells for hepatitis B virus. Culture medium is withdrawn at 6 hour intervals and tested for human antibodies specific for hepatitis B virus using well known immunological methods. When anti-hepatitis B virus antibodies are detected, the human PBL are isolated from the chimeric organ and fused to make hybridomas that produce human monoclonal antibodies specific for hepatitis B virus-infected cells using fusion methods well known to those skilled in the art including those described in Olsson and Kaplan, Proc. Natl. Acad. Sci. U.S.A. 77:5429, 1980; Chiorrazzi et al., J. Exp. Med. 156:930, 1982; Kozbor et al., Proc. Natl. Acad. Sci. U.S.A. 79:6651, 1982; and Wasserman et al., J. Immun. Methods 93:275-283, 1986.

Example 4 Chimeric Tissue Containing Target Lymphocytes and Dendritic Cells

The method used is essentially as described in Example 1 for chimeric organ tissue containing target lymphocytes with the following changes. The dose of irradiation is changed to supralethai dose (greater than 300 rads) to kill all immune cells. The source of immune cells is bone marrow rather than blood. Dendritic cells and/or other cells present in bone marrow can be isolated prior to introduction into the organ tissue by sorting with a FACS cell sorter using standard methods. The human bone marrow cells can be immunized using immunogens well known in the art and methods essentially as described in Examples 1-3.

Example 5

Production of Human Monoclonal Antibodies Using Chimeric Organ Immunization to Stimulate Antigen-specific B Cells A chimeric organ made up of mouse splenic tissue and human immune system target cells is made essentially as described in Examples 1 and 4. The organ is immunized in vitro using the culture conditions essentially as described in Example 1, with an immunogen including cholera toxin B subunit (CTB)) at 1-10 μg/ml of culture medium or concentrations easily determined by those skilled in the art using routine testing. After 6, 12 and 24 hours, aliquots of culture medium are withdrawn from a resealable sampling port of an automated culture system essentially as shown in FIG. 1. The culture medium is tested for the presence of human antibodies specific for CTB using well known immunological methods including ELISA and RIA. When human anti-CTB antibodies are detected, the chimeric organ is removed from the culture system and human cells are isolated using well known techniques (e.g., fluorescence activated cell sorting). The isolated human ceils are then fused to human lymphobiastoid cell lines and thereafter maintained in culture medium selective for hybridomas using techniques well known in the art as described in Olsson and Kaplan, Proc. Natl. Acad. Sci. U.S.A. 77:5429, 1980; Chiorrazzi et aL, J. Exp. Med. 156:930, 1982; Kozbor et al., Proc. Natl. Acad. Sci. U.S.A. 79:6651, 1982; and Wasserman et al., J. Immun. Methods 93:275-283, 1986. After growth of the monoclonal hybridomas in culture, the culture supernatants are tested immunologicaliy for the production of human anti-CTB antibodies as described above.

Example 6 In Vitro Cell Immunization to Produce Anti-tumor Antibodies

Tumor cell antigens in the context of MHC molecules (class I or class II) are isolated from cultured tumor cell lines or biopsy tumor tissue (e.g., mastocytoma, thymoma, lymphoma, melanoma, hepatoma) by lysis of the cells and standard methods to purify cell plasma membrane components (e.g., centrifugation and/or affinity chromatography). Although this example specifically presents in vitro cell treatment to produce anti-colon cancer antigen (e.g., CEA) antibodies, it will be understood that any of a variety of well-known (e.g., CA19-9 or σ-fetal protein) or easily discoverable tumor antigens could be used in production of anti-tumor antibodies. Indeed, the tumor antigen used to stimulate cells in vitro does not need to be specifically identified because isolated cell plasma membrane components from any tumor cell line or tumor tissue may be used as the stimulatory immunogen in vitro.

A human colon cancer cell line (e.g., COLO 201 or any other colon cancer cell line generally available from the American Type Culture Collection, Rockville, Maryland) is grown in vitro and plasma ceil membrane fractions are isolated using standard techniques. Purified plasma membrane fractions are suspended in Modified Waymouth's MB medium at 1-1,000 μg/ml.

A chimeric organ comprising rat spleen tissue and human lymphocytes is made essentially as described in Example 1. The chimeric organ is cultured in the automated culture system essentially as shown in FIG. 1 and described in Example 1. To the culture medium in the automated culture system, 5-20 μg of purified colon cancer plasma membrane is added and the cells are cultured for 12-36 hours. Samples of culture medium are removed at 6 hour intervals and tested for the presence of anti-CEA antibodies using standard methods (e.g., immunoprecipitation or immunofluorβsceπt/i? situ binding to cultured cells used to produce the immunogen). When newly produced human antibodies are detected, the human lymphocytes in the chimeric organ are recovered and used in standard fusion methods to produce a human hybridoma for production of anti-tumor antibodies. Example 7

In Vitro Cell Immunization to Produce Anti-cell Antigen Antibodies Cell antigens, including MHC class I and class II antigens and accessory glycoproteins on the surface of cells, can be used as immunogens for in vitro cell treatment of human cells in chimeric organs. The antigens are prepared from cultured human cell lines using standard affinity chromatography methods well known in the art. This example uses CD3 glycoprotein, expressed by all types of T cells, as the immunogen but those skilled in the art will appreciate that any cell antigen could be substituted as an immunogen.

CD3 glycoprotein is isolated from cultured human T cells or T cells isolated from whole blood using standard methods and affinity chromatography with an anti-CD3 antibody. The purified CD3 molecules are suspended in a physiological salt solution at a concentration of 1-100 μg/ml. A chimeric organ comprising rat spleen tissue and human lymphocytes is made and cultured in the automated culture system essentially as described in Example 1. One to 20 μg of the purified CD3 immunogen is added to the culture medium and culture conditions are maintained for an addition 24-48 hours with the immunogen. Culture medium aliquots are removed from the resealable sampling port at 24, 36 and 48 hours after CD3 treatment began and samples are tested for the presence of anti-CD3 antibodies using standard immunoprecipitation methods compared to a known anti-CD3 antibody. When newly produced human anti-CD3 antibodies are detected, the human lymphocytes in the chimeric organ are recovered and fused using standard methods to produce a human hybridoma f o r p r o d u c t i o n o f a n t i - C D 3 a n t i b o d i e s .

Example 8 Animal Model for Testing Products Made from In Vitro Cell Treatment To facilitate testing of antibodies and/or cells produced by the in vitro cell treatment (IVCT) methods as disclosed in Examples 1-7, an animal model for testing the efficacy of these products was also needed before the antibodies or cells could be used in producing a medicament for treating humans. An animal model of a human infection for testing human monoclonal antibodies or IVCT products was produced by infecting a human virus into an animal under conditions where a viral infection was maintained for a period of 1 -10 days, during which time the IVCT products can be tested in situ. Twenty-one mixed breed white rats were infected human plasma obtained from a patient having an active hepatitis B virus (HBV) infection (1,750 pg/ml of HBV DNA in the plasma). Alternatively, infection of the rat may be initiated with injection of about 250 mg of human tissue (e.g., minced tissue or in a single cell suspension of infected human liver) per kg body weight, although this method was not used here to prevent recovery of cccDNA from the injected human tissue (see below). At one day before inoculation with the human HBV-containing plasma, the rats received a partial hepatectomy (about 50% to 80%) to enhance mitosis and, presumably, to increase permeability of nuclear membranes and sinusoidal endothelial walls. Generally, about 75% of the liver tissue was removed about 24 before infection. Splenectomy or a combination of splenectomy and partial hepatectomy may also be used. For inoculation, the rat was anesthetized, surgically opened and inoculated by injecting the human plasma directly into the liver parenchyma and then the rat was surgically closed. Inoculation by intravenous, intraperitoneal and portal vein injection was tried in other experiments in which the results were not as reproducible as parenchyma inoculation.

To allow cross-species infection of human HBV into the rat liver tissue, the rats were treated an immunosuppressant drug, methylprednisolone sodium succinate (10 ^s M/kg/day) to suppress the immune response, beginning about 1 day before infection. Other immunosuppressants (e.g., FK 506 and cyclophosphamide) were used in other experiments. Three animals were sacrificed daily for analysis of viral infection (e.i., for viral DNA and antigens) until the seventh day after inoculation. During the post-infection period, rat plasma was collected from the animals' tails and tested for the presence of HBsAg and/or the HBV envelope antigen (HBeAg) using well known immunodetection methods for these antigens (e.g., using commercially available reagents available from Abbott Diagnostics, Chicago, IL), with a signal-to-noise ratio of higher than 2.1 considered positive (Gripon P. et al., Virology 192:534-540, 1993).

HBV replication in humans produces distinct DNA species arising from conversion of the HBV genomic DNA (relaxed circular double-stranded DNA of about 3 kb) into a covalently closed circular DNA (cccDNA) of about 2.1 kb. The minus strand of the cccDNA is transcribed into mRNA of 2.1 and 2.4 kb and pregenomic RNA of 3.4 kb. The pregenomic RNA is the template for production of a complementary minus-strand DNA which is followed by production of the plus strand. The existence of these various forms of HBV DNA species was assayed using extrachromosomal DNA extracted from the animal livers (fractionation as described in detail in Hirt, B„ J. Mol. Biol. 26:365-369, 1967). Briefly, liver tissue was lysed in 10 mM Tris-HCl, pH 8.0, 1 % SDS, 100 mM NaCl, 10 M EDTA and 0.1 mg/ml proteinase K for 12 hr at 50°C; then the lysate was incubated with 1 M NaCl, overnight at 4°C; and then cellular DNA was pelleted at 10,000 * g for 1 hr and the supernatant was deproteinized by phenol- chloroform extraction. The DNA in the supernatant was precipitated with ethanol, dissolved in 10 mM Tris-HCl, pH 8.0 and 1 mM EDTA and analyzed by Southern blotting (Southern E.M., J. Mol. Biol. 98:503-517, 1975) with the DNA immobilized on a nylon membrane. The immobilized DNA was probed with a ³²P-labeled HBV DNA probe cloned into pBR322. As a positive control, human plasma containing 1,750 pg/ l of HBV DNA was included in the Southern blot.

The results of the Southern blot analyses showed that all of the animals sacrificed on day three post- infection and one of the animals sacrificed on day two post-infection had a 2.1 kb band of DNA that hybridized to the HBV probe, this size being consistent with cccDNA normally seen in HBV replication in humans. These same rats also showed 3.2 kb and 1.5 kb bands of DNA that hybridized with the HBV probe. None of these bands were detected in rats sacrificed after day three post-infection. The positive control human patient serum contained bands of 3.2 kb and 2.1 kb. These results show that HBV replication occurred in the infected rats, producing similar DNA species as seen in an infected human. The kinetics of DNA replication in the rats were similar to results seen in cultured infected cells (Gripon P. et al., Virology 192:534-540, 1993; Bchini R. et al., J. Virol. 64:3025-3032, 1990; Ochiya T. et al., Proc. Natl. Acad. Sci. USA 86:1 B75-1879, 1989).

The HBsAg was found in the plasma of all infected animals until day four post-infection with the average amount (signal/noise ratio) detected being: 56.4 on day one, 24.2 on day two, with similar amounts detected until day four but only minimal positive values detected on day five post-infection. The HBeAg was found in one-third of the infected animals at day three and in two-thirds of the animals at day four post-infection. Although the average HBeAg amounts detected were more variable than for the HBsAg, a similar trend of detectable HBeAg diminishing by day five post-infection was observed. These results are also consistent with other studies of viral replication in the infected rats in that the antigens appeared with infection and progressively decreased after detection of cccDNA (Gripon P. et al., Virology 192:534-540, 1993; Bchini R. et al., J. Virol. 64:3025-3032, 1990; Ochiya T. et al., Proc. Natl. Acad. Sci. USA 86:1875-1879, 1989; Galle P.R. et al., Gastroenterol. 106:664-673, 1994).

In other experiments, rats were similarly infected and monitored for the presence of HBV antigens either when the rat died or, for those rats that survived, at 11 days post-infection when the rats were killed and assayed. Various rat tissues were prepared and examined for the presence of HBsAg and HBeAg using histological surface staining with fluorescent-tagged anti-HBsAg and anti-HBeAg antibodies. These assays showed the presence of HBsAg in the liver tissue but not in kidney, thymus or pancreas between one to six days post-infection but the infection was not detected by the ninth day post infection. Where surface staining was detected, fluorescent-tagged anti- HBsAg antibody stained the liver tissue in discrete spots in the sectioned tissue using usual pathological detection methods indicating foci of infection. Essentially the same time course was observed for HBeAg which was found in the blood serum at 1-6 days post-infection but was not detected at 11 days post-infection.

This animal model of human HBV infection can be used to test the efficacy of antibodies and IVCT products as preventive or treatment compositions. For example, for testing preventive effects, anti-HBV monoclonal antibodies can be injected into the rat 1-24 hours before inoculation with HBV and then the rats can be killed and their level of HBV infection monitored immunohistologically or by other immuπodetection methods daily for 1-11 days post- infection. This animal model can be used to monitor the IVCT of chronic viral infection with immunological tolerance by immunizing the animal cells in vitro or by killing the virus in vitro and then infusing the target cells back into the animal or person from which the target cells were isolated. Alternatively, the animal model can also be used to monitor the treatment of a HBV infection by injecting i.v. human monoclonal anti-HBV antibodies into an infected animal at 648 hours post-infection and then monitoring the level of infection daily as described above. I vJ be appreciated by those skilled in the art that transgenic mice that are immunotolerant to the virus could similarly be infected with a large dose of virus thus eliminating the need to use immunosuppressant drugs or splenectomy to limit the animal's immune response to the virus.

Claims

I CLAIM:

1. A method of treating cells in culture comprising the steps of: providing organ tissue from a first animal to serve as a host tissue in in vitro culture; providing target cells from a second animal; incubating said host tissue with said target cells to produce a chimeric organ; and culturing said chimeric organ in vitro.

2. The method of Claim 1, wherein said target cells are immune system cells.

3. The method of Claim 2, wherein said immune system cells are lymphocytes, dendritic cells or a mixture thereof. 4. The method of Claim 2, further comprising irradiating said host tissue with a sublethal dose of irradiation to kill resident lymphocytes before said target immune system cells are incubated with said host tissue.

5. The method of Claim 2, further comprising irradiating said host tissue with a supralethal dose of irradiation to kill resident immune cells before said target immune system cells are incubated with said host tissue.

6. The method of Claim 1, wherein said host tissue is spleen tissue. 7. The method of Claim 1, wherein said first animal is selected from the group consisting of rats, mice, dogs, chickens, frogs and humans, and said second animal is selected from the group consisting of rats, mice, dogs, chickens, frogs and humans.

B. The method of claim 7, further comprising the steps of treating in vitro target cells from a human having a pathologic condition and returning target ceils to a human having a pathologic condition after treatment of said target cells in vitro.

9. The method of Claim 2, further comprising immunizing said target immune system cells by exposing said chimeric organ to an immunogen in in vitro culture.

10. The method of Claim 9, wherein said immunogen is an antigen selected from the group consisting of human cell antigens, tumor antigens and viral antigens. 11. The method of Claim 10, further comprising collecting said target cells from said chimeric organ for transplantation of said target cells into a recipient mammal.

12. The method of claim 9, further comprising testing said target immune system cells in an animal model of a human disease.

13. A method of immunizing cells in culture comprising the steps of: providing organ tissue from a first animal to serve as a host tissue in in vitro culture; providing target immune system cells from a second animal; incubating said host tissue with said target immune system cells to produce a chimeric organ; and immunizing said target cells in said chimeric organ by exposing said chimeric organ to an immunogen in vitro. 14. The method of Claim 13, further comprising irradiating said host tissue with a sublethal dose of irradiation to kill resident lymphocytes before said target immune system cells are incubated with said host tissue. 15. The method of Claim 13, further comprising irradiating said host tissue with a supralethal dose of irradiation to kill resident immune cells before said target immune system cells are incubated with said host tissue.

16. The method of Claim 13, wherein said immunogen is an antigen selected from the group consisting of human cell antigens, tumor antigens and viral antigens. 17. The method of Claim 13, wherein said first animal is selected from the group consisting of rats, mice, dogs, chickens, frogs and humans and said second animal is a human.

18. The method of Claim 13, further comprising removing said target immune system cells from said chimeric organ after immunizing said target cells and fusing said target cells with cells capable of producing a hybridoma for production of monoclonal antibodies. 19. The method of Claim 18, further comprising producing said monoclonal antibodies from said hybridoma.

20. Monoclonal antibodies for treating a pathological condition in humans produced by the method comprising the steps of: providing organ tissue from an animal to serve as a host tissue in vitro; providing target immune system cells from a human; incubating said animal host tissue with said human target cells to produce a chimeric organ; immunizing said human target cells in said chimeric organ by exposing said chimeric organ to an immunogen in vitro; fusing said human target cells with cells capable of forming a hybridoma for production of monoclonal antibodies; and producing human monoclonal antibodies from said hybridoma.

21. The monoclonal antibodies of Claim 20, wherein said human monoclonal antibodies specifically bind to an immunogen selected from the group consisting of human cell antigens, tumor antigens and viral antigens.

22. A mammal infected with a human virus produced by the steps comprising: treating a mammal with an immunosuppressant drug, partial hepatectomy, splenectomy or any combination thereof; infecting said treated mammal with a human virus within 24 hours after treatment; and maintaining said infected mammal alive for one to eleven days after infection.

23. The mammal of Claim 22, wherein said human virus is a hepatitis virus. 24. The mammal of Claim 22, wherein said mammal is a transgenic mouse that is tolerable to said virus.

25. The mammal of Claim 22, wherein said mammal is a fetal mammal.