NO885016L - PROCEDURE FOR THE PREPARATION OF ANTIGEN-SPECIFIC T-CELL LINES AND THERAPEUTIC APPLICATION OF THESE. - Google Patents

Description

The application is a Continuation-in-Part of US patent application no.

933,789 filed November 24, 1986.

The invention described here was carried out with assistance

from National Institutes of Health Grant No. 1-P01-HD19937-01A1. The United States has certain rights in this invention.

Although the mechanisms and agents involved in the immune response in mammals are not fully understood, techniques for manipulating the immunological response have great therapeutic potential. This is particularly evident in the treatment of pathological conditions which lead to suppression of the normal immune responses and which cannot be so easily treated by usual drug-based therapies. Such conditions include certain viral infections and the immunosuppression caused by the administration of anticancer drugs, drugs used to treat autoimmune diseases

and those used to prevent rejection of organs after transplantation.

The ability to respond to immunological stimuli lies mainly

in the cells of the lymphatic system. During embryonic life, a stem cell develops that develops differently along several different lines. E.g. a stem cell can be transformed into a lymphoid stem cell that can develop differently to form at least two distinct lymphocyte populations. One population called T-lymphocytes is the eliciting agent (effector) in cell-mediated immunity, while the other (B-lymphocytes) is the main elicitor of antibody-regulated or humoral immunity. The stimulus for B-cell antibody production is the attachment of an antigen (Ag) to B-cell surface immunoglobulin. Thus, B cell populations are mainly responsible for specific antibody production, (Ab) production, in the host. Sometimes, and for certain antigens (Ags), B cells require cooperation with T cells for effective Ab production.

Of the classes of T lymphocytes, T helper cells (T^ cells) are antigen-specific cells involved in primary immune recognition and host defense reactions against bacterial, viral, fungal and other antigens. The cytotoxic cells, (Tc) cells, are antigen-specific effector cells that can kill target cells after they are infected with pathological agents.

Although T helper cells (Tn cells) are antigen specific, they cannot recognize free antigen. In order for recognition and subsequent Tft cell activation and proliferation to take place, the antigen must be presented to recipients or a recipient complex on the T^ cell together with the most important (major) products of the histocompatibility complex (histocompatibility complexMHC) class II, or "leukocyte antigens". Thus, T^ cell recognition of pathogenic antigens is MHC class II "restricted", in that a given population of T^ cells must either be autologous or share one or more of the limiting leukocyte antigen (LA) specificities expressed by the MHC on the host. Similarly, Tc cells recognize Ag in association with class I MHC LAs.

In the case of T^ cells, this function is performed by a limited number of specialized cells called "antigen presenting cells" (APC). It is now generally recognized that T helper cells (Tft cells) recognize processed soluble antigen in association with class II MHC LA expressed on the surface of macrophages.

More recently, other cell types, such as resting and activated B cells, dendritic cells, epidermal Langerhans cells and human dermal fibroblasts have also been shown to present antigen to T cells. Epstein-Barr virus-transformed human lymphoblastoid B cells (LCL) have been shown to present tetanus toxoid, M. leprae, and Candica albicans to autologous antigen-specific T cells.

If a given T^ cell has receptors or a receptor complex that enables it to recognize the MHC class II LA antigen complex, it is activated, multiplies and produces lymphokines such as interleukin 2 (IL-2). The lymphokines in turn cause the proliferation of a number of types of "killer cells", including T cells and macrophages, which can exhibit antimicrobial and tumoricidal activity. After the stimulation subsides, those that survive the expanded T^ cells remain as memory cells in the body and can rapidly expand again when the same antigen is presented. The importance of T cells in healing from acute viral infections is well recognized for certain viruses, in particular the myxovirus (influenza), smallpox viruses (ectromelia and vaccinia)

and the arenavirus (lymphocytic choriomeningitis virus).

Despite the fact that the importance of the immune system's defense mechanisms for the well-being of the host has been clearly demonstrated, the therapeutic potential of these agents has not been realized. In a preliminary investigation, S.A. reported Rosenberg et al., New England J. Med.. 313, 1485 (1985) that the systemic administration of autologous peripheral blood lymphocytes (PBL) that had been incubated with IL-2 together with additional IL-2 to patients with advanced cancer achieved cancer regression in 11 of the 25 patients treated. Rosenberg et al. termed the incubated PBL cells "lymphokine-activated killer (LAK) cells" and reported that they were members of a cytolytic system distinct from natural killer cells and cytotoxic T cells. However, later studies where this therapy was used have not confirmed these promising early results.

Regardless of the long-term results of this method of "adoptive immunotherapy", Rosenberg et al. noted that "the major difficulty in applying this method to the treatment of human cancer has been the inability to generate sufficient numbers of autologous human cells with antitumor reactivity that can be used for systemic therapy". This is particularly problematic regarding patients who are immunosuppressed due to infections, cancer, drugs in connection with organ transplantation or antineoplastic drugs and the like.

Likewise, a number of attempts have been made to isolate and maintain homogenous populations of Tc or T₂ cells and to characterize them by their antigen specificity and MHC restriction. These experiments usually involve stimulation of mononuclear cells from a seropositive human or mouse host with bacterial or viral preparations in combination with non-proliferative (non-profilerative) APCs, such as irradiated autologous mononuclear cells (MNCs). Proliferating polyclonal populations of T₂ cells or Tc cells are cloned by limiting dilution to obtain homogeneous populations and then further propagated and characterized by a variety of techniques. As stated by Rosenberg et al. in the case of cloned LAK cells, one of the major obstacles to T-lymphocyte cloning is the limited availability of autologous or alternatively allogeneic MHC LA-matched MNCs, particularly from clinical subjects.

To overcome this problem, APCs other than autologous MNCs have been used as APCs. E.g. have T. Issekutz et al.,

J. Immunol., 129, 1446 (1982) first indicated that autologous Epstein-Barr virus-transformed (EBV-transformed) LCL lines can present antigens associated with tetanus toxoid to tetanus-reactive polyclonal T cells and T cell clones. D.R. Kaplan et al. in Cellular Immunolo<g>y , 88., 193 (1984) reported the production of three Tc cell clones by the propagation of peripheral blood MNCs from a type A influenza immune donor. One of the clones proliferated in the presence of irradiated virus-infected autologous MNCs, or in the presence of irradiated infected Epstein-Barr virus-transformed allogeneic lymphoblastoid cells (LCL).

B. G. Elferink et al., Scand J. Immunol., 22, 585 (1985) further demonstrated that autologous and allogeneic Epstein-Barr virus-transformed (EBV-transformed) LCL lines can present antigens associated with M. leprae bacilli to M. leprae -reactive cloned T cell lines. Another paper from this group described the isolation of a T-cell clone that can recognize only M. leprae antigens. The cloning method used autologous EBV-transformed LCLs, as APCs. The advantage of using EBV-transformed LCLs is that they can provide a continuous and unlimited source of APCs. [J.B.A.G. Haanen et al., Scand. J. Immunol., 23, 101 (1986)].

Human cytomegalovirus (HCMV) is a large species-specific herpesvirus (DNA 1.5x10^ Da) that shares the characteristics of latency and reactivation with other members of this group. HCMV is ubiquitous (30-70% of adults are seropositive), but the main site or sites of latency of HCMV have not been determined, nor are the molecular events involved in its reactivation. HCMV infection and reactivation can be asymptomatic but are associated with significant morbidity and mortality in immunocompromised individuals. E.g. HCMV is the most common cause

opportunistic infection in bone marrow transplant patients.

More than 80% of those who develop HCMV pneumonia die, despite the latest treatment methods.

Although monoclonal antibody therapy may be useful as a preventive treatment, it has been suggested that survival of immunosuppressed patients with severe HCMV infections may depend on the presence of HCMV-specific cytotoxic T cells. Quinnan et al., in New England J. Med., 307, 7 (1982) have suggested that HCMV-specific cytotoxic T-cell responses in these patients are associated with clinical cure and cessation of virus excretion, whereas those who had active infections that did not developed cytotoxic responses all of whom died.

R. C. Gehrz et al., Lancet, 2, 844 (1977) reported that infants with congenital HCMV infection have an antigen-specific defect in T helper cell proliferation, which is associated with persistence of replicating viral infection for months and years, despite the presence of HCMV-specific antibodies. Acquisition of HCMV-specific lymphocyte proliferative responses appears to be associated with an impairment in virus excretion. Thus, it is likely that HCMV-specific T helper cells play a significant role in the immune defense against this virus in immunocompetent hosts.

In patients with reactivated HCMV infection, antibodies directed against HCMV antigens are not protective or are only useful in the context of appropriate T-cell responses. Peripheral blood lymphocytes (PBL) are also unlikely to be useful as therapeutic agents, even if they could be obtained in sufficient quantities. The number of T helper cells or cytotoxic T cells reactive with a particular antigen is extremely low and thus the selective expansion of antigen-specific T cells in vitro will be necessary to obtain sufficient numbers for therapeutic purposes. Furthermore, the therapeutic administration of allogeneic PBLs will activate T cells that recognize "foreign" leukocyte antigens, leading to a mixed leukocyte culture reaction. This mixed leukocyte culture reaction can activate unwanted non-specific immune responses, or alternatively induce suppressor cells that can inhibit desired antigen-specific

immune responses.

Autologous or allogeneic antigen-specific T cell lines are likely to express a desired therapeutic activity without attendant complications associated with the mixed leukocyte culture reaction. L. K. Borysiewicz et al., Eur. J. Immunology, 13, 804 (1983) reported the generation of short-lived polyclonal T₂ cell lines by the expansion of MNCs from seropositive subjects with soluble HCMV antigen in the presence of IL-2. When the MNCs were co-cultured on autologous HCMV-infected fibroblasts, polyclonal Tc cells were generated, which lysed HCMV-infected cells.

However, a need exists for improved methods of generating and maintaining populations of T₂ cells and Tc cells specific for antigens associated with viruses such as HCMV and other pathogenic agents. A further need exists for immunotherapeutic methods based on the delivery of antigen-specific T-lymphocyte populations of known biological activity to mammalian subjects.

Brief description of the invention

I. Production of T-cell lines

A. T helper cells

The present invention is directed to methods for the efficient production of a homogeneous population of T helper cells (Tft cells) which are specific for a viral antigen such as an HCMV antigen. It is believed that the present methods provide the basis for the efficient production of cloned T cells, using a minimum of antigen and lymphokine support while maintaining both the antigen specificity and the functional activity of the clones. In its broadest form, the present method comprises: (a) isolating a population of mononuclear cells (MNC) from the blood of a donor mammal, wherein the MNC population comprises T helper cells specific for a viral antigen, and autologous, non- proliferating antigen-presenting cells (APC);

(b) combining said isolated population of said MNCs with an amount of the viral antigen that is effective

to effect the proliferation of a T helper cell specific for the antigen; (c) allowing said T helper cell to proliferate for a period of time effective to allow non-proliferating MNCs to lose viability, and

(d) clonally expanding the antigen-specific T helper cell

in the presence of a plurality of non-proliferating antigen-presenting cells comprising a mixture of (i) autologous MNCs, allogeneic MNCs or mixtures thereof, and (ii) autologous lymphoblastoid cells (LCLs), allogeneic LCLs or mixtures thereof, and a quantity of the said viral antigen which is effective for the propagation of the said cloned antigen-specific T-helper cell to give the said homogeneous population, and wherein the said LCLs are present in an amount which is effective for increasing the rate of propagation of the T-helper cell in relation to that effected of the MMCs.

During the selective propagation step (c), which may require 1-2 weeks, it may also be effective to add a further amount of said viral antigen and the autologous non-proliferating APCs to effect the further propagation of the T helper cell for to enhance the production of a viable polyclonal population of the virus antigen-specific T^ cells prior to step (d). In step (d), antigen-specific monoclonal T₂ cells can be obtained by limiting dilution or by single cell isolation by micro-manipulation or flow cytometry.

Antigen-specific polyclonal T cell lines, as well as monoclonal antigen-specific T cells, have potential therapeutic utility. Thus, the term "T cell line", as used herein, can refer to an expanded population of polyclonal T cells that are uniquely reactive with a given antigen, or to a homogeneous population of monoclonal T cells that has been obtained by

the expansion from a single precursor T cell.

As the present invention includes the propagation of antigen-specific T-cells, it also provides a method for the propagation of a homogeneous population of T-helper cells specific for viral antigen comprising combining said homogeneous population of T-helper cells with an amount of the antigen and an amount of a mixture of non-proliferating antigen-presenting cells (APCs) effective to effect the proliferation of the population of T-helper cells, said mixture of APCs comprising (i) autologous or allogeneic lymphoblastoid cells (LCL) and (ii ) autologous or allogeneic mononuclear cells (MNC), in that the LCLs are present in an amount that is effective for increasing the multiplication rate of the T-helper cells in relation to what is effected by the MNCs. Preferably, the LCLs are obtained from a continuous lymphoblastoid cell line, such as that which can be produced by viral transformation, hybridoma technology and the like. The APCs are made non-propagating by techniques known in the art, e.g. by X-ray irradiation, treatment with chemical agents such as mitomycin C and the like.

Both the nonproliferating MNCs and LCLs have been found to induce the proliferation of T^ cells when obtained from the same host as the T cells or are allogeneic MNCs or LCLs that share one or more class II restriction antigens (abbreviation: "MHC LA -matched"). It was surprisingly found that a mixture of LCLs and MNCs can synergistically present antigen to T^ cells, thus significantly increasing their proliferation rate compared to that effected by an equivalent number of one or the other type of APC when the used alone.

For example, this effect is observed in the case of a viral antigen such as an HCMV antigen, when a mixture of EBV-transformed autologous LCLs and autologous MNCs is used as the APCs in a ratio of at least 1:10. Preferably, the ratio of T cells to LCL cells added is from 1:1 to 1:3, and the ratio of T cells to MNCs is from 1:3 to 1:10.

Furthermore, only a limited density of LCL is required to significantly increase the expansion of T^ clones. This aspect of the present method is important from a practical point of view, because human antigen-specific T₂ clones can be expanded in large quantities in a relatively short period of time using limited numbers of MNCs. E.g. according to the present method, HCMV-specific T^ clones can be expanded from 1-2xl0<6> cells to 10^ cells within 2 weeks using 1-2xl0<7>MNC and lxl0<6>LCL as APC.

It has also been found that it is far preferable to carry out propagation steps (c) and/or (d) of the method in the presence of an effective amount of interleukin-2 ("IL-2" or "TCGF").

A further purpose of the present invention comprises increasing the multiplication rate of the virus antigen-specific T^ cells by carrying out the original propagation step (steps a-c as indicated above), or the subsequent expansion step (step (d), as indicated above) in the presence of a amount of monoclonal antibody specific for said virus antigen. In one embodiment of this aspect of the invention, the rate of proliferation of a T^ cell specific for an HCMV viral antigen is increased by combining MNCs isolated from the blood of an HCMV-seropositive donor mammal with a monoclonal antibody specific for a antigen present on an HCMV gene product, such as a structural protein. The monoclonal antibody is used in combination with (a) an effective proliferation-stimulating amount of said antigen and (b) an amount of autologous APCs selected from the group consisting of (i) non-proliferating MNCs, (ii) a non-proliferating LCL obtained from a continuous LCL and (iii) mixtures thereof, e.g. mixtures which together may act to further increase the proliferation rate of the T^ cells. Preferably, the autologous, non-proliferating antigen-presenting cells are selected from the group consisting of X-rayed autologous MNCs, a X-rayed Epstein-Barr virus-transformed (EBV-transformed) lymphoblastoid cell line (LCL), and mixtures thereof. These are the preferred APCs for use in step (b) of the method, regardless of whether or not monoclonal antibody and/or IL-2 is used to increase the proliferation rate.

B. C<y>totoxic T cells

The present method can also be used to produce a homogeneous population of cytotoxic T cells (Tc cells) specific for an HCMV antigen. This aspect of the present invention comprises the following steps: (a<1>) isolating a population of mononuclear cells (MNC) from the blood of a mammalian donor, preferably a human,

wherein the MNC population comprises Tc cells specific for said viral antigen,

(b<1>) combining said isolated population of said MNCs with (i) a quantity of an autologous, non-proliferating, antigen-presenting cells (APC), e.g. irradiated MNCs, (ii) an amount of said HCMV antigen and (iii) an amount of interleukin-2 (IL-2) effective to cause the proliferation of a Tc cell specific for said antigen;

(c<1>) allowing said Tc cell to proliferate for a period of time effective to allow non-proliferating MNCs to lose viability; and

(d<1>) to clonally expand said antigen-specific Tc cell

in the presence of (i) a plurality of non-proliferating, autologous, antigen-presenting cells; non-proliferating, allogeneic, antigen-presenting cells or mixtures thereof, (ii)

an amount of IL-2 and (iii) an amount of said HCMV antigen effective to propagate said cloned antigen-specific Tc cell to yield said homogeneous population.

Step (b<1>), (c<1>) or (d<1>) is preferably carried out in the presence of a monoclonal antibody which is specific for an HCMV antigen and which serves to increase the proliferation rate of said Tc cell .

As previously described for antigen-specific T helper cell lines, polyclonal Tc lines reactive with a particular viral antigen can be expanded by continuous stimulation of Tc blasts that exhibit cytotoxic activity against target cells expressing the desired antigen. Homogeneous populations of virus-specific Tc clones obtained from a single progenitor T cell can be obtained by limiting dilution or by single cell isolation by micromanipulation or flow cytometry, followed by expansion according to step (d<1>).

Other preferred embodiments of this Tc cell propagation method include (1) the use of whole HCMV virus antigen or the use of HCMV-infected autologous or allogeneic fibroblasts as a source of cell-associated virus antigen, e.g. an HCMV intermediate-early protein, (2) the addition of additional amounts of the viral antigen IL-2 and the autologous non-proliferating APCs during step (c<1>) to effect further proliferation of said Tc cell and (3 ) the use, in step (b<1>) or step (d<1>) of antigen-presenting cells further comprising non-proliferating MNCs obtained from a continuous autologous MNC line in an amount effective to increase the proliferation rate of Tc -cells compared to that effected by the autologous mononuclear antigen-presenting cells. This continuous autologous MNC line preferably comprises a virus-transformed LCL, i.e.

an EBV-transformed LCL.

The present invention is also directed to a method for propagating a homogeneous population of Tc cells which is specific for an antigen, such as a viral antigen, i.e. an HCMV antigen. The method comprises combining said homogeneous population of cytotoxic T cells with an amount of said antigen, an amount of interleukin-2 (IL-2) and an amount of a mixture of non-proliferating antigen-presenting cells (APC) which are effective to effect the propagation of said population of Tc cells, said mixture of APCs comprising allogeneic virus-transformed lymphoblastoid cells (LCL) and allogeneic mononuclear cells (MNC). The method further comprises combining said population of Tc cells with an amount of monoclonal antibody specific for said antigen, which monoclonal antibody serves to increase the proliferation rate of said population of cells.

The term "antigen" as used herein with respect to a pathological target, such as a virus or an infected cell, refers to a compound such as a polypeptide, polypeptide complex, glycoprotein, nucleic acid or the like that produces an immune response. A pathological target antigen can be a portion of the pathological target itself, e.g. a viral envelope glycoprotein, or it may be an antigen expressed by a diseased tissue, such as a neoplastic tissue or a virus-infected cell. An "antigen-specific" T-lymphocyte is activated in the presence of a single antigen when presented to an antigen-presenting cell (APC) in conjunction with a specific leukocyte antigen (LA) expressed by the antigen-presenting cell. An antigen-specific T^ cell will proliferate in a suitable medium in the presence of the antigen for which it has specificity when said antigen is presented to the T^ cell by an APC which also expresses the leukocyte antigen for which the T^ cell has specificity. As indicated above in the case of human T helper cells, the specific leukocyte antigen will be a human MHC class II antigen, whereas human cytotoxic T cells are specific for MHC class I antigens.

C. HCMV antigen-specific T cell lines

Preferred embodiments of the present invention comprise homogeneous populations of Tc or T^ cells and methods for their production, where a given cell population is specific for an HCMV antigen.

When a T cell or a T cell clone is said to be antigen specific, the epitope or the specific antigen site on the antigen mixture gene is not necessarily known, although the mixture comprising the antigen may be specified. E.g. three different homogeneous populations of T helper lymphocytes can be specific for one particular HCMV structural protein or glycoprotein. Although they all respond to the same antigen and thus have the same antigen specificity, they may have different specificities at the submolecular level, meaning that they may respond to different regions of the antigen or epitopes. Within this context, different HCMV-specific T lymphocytes may have the same submolecular specificity only when they recognize the same epitope on the same HCMV-associated antigen. However, they can still be specific for the same antigen if the antigen has a number of different epitopes that are presented by APCs in different cases.

E.g. the HCMV DNA genome is transcribed in sequential order, beginning with the restricted transcription of intermediate-early genes that encode regulatory proteins necessary for the subsequent expression of early genes. The major intermediate-early gene (I-El) encodes a 68 kD regulatory protein that is expressed on the membrane of infected cells 6-24 hours after infection has begun. HCMV-specific non-cytotoxic T cells are thought to mainly recognize this intermediate-early protein as part of their role in immune surveillance to prevent reactivation of latent HCMV.

Transcription of early genes precedes the beginning of DNA synthesis. Included among the early gene products are virus-specific polymerases and kinases that are necessary for DNA replication. These enzymes do not seem to play an important role in immune responses.

However, the late HCMV genes encode a number of immunogenic structural proteins and glycoproteins. Included among these proteins are envelope glycopeptide complexes with disulfide bridges, non-glycosylated envelope proteins, tegument glycoproteins that form a bridge between the viral nucleocapsid and the outer envelope, and matrix proteins that form the internal capsid structure of the virus.

The purification and characterization of a variety of immunogenic structural glycopeptide complexes and reduced glycopeptides from the HCMV envelope fraction along with monoclonal antibodies (MoAber) specific thereto is fully described in US Patent Application No. 933,789 filed November 24, 1986, which is hereby incorporated by reference. The primary glycopeptides, glycopeptide complexes and MoAbers described are listed in Table 1.

The immunogenic glycopeptide complexes and glycopeptides listed in Table 1 are useful for selectively stimulating the proliferation of T₂ cells obtained from the blood of an HCMV seropositive donor. In the same way, monoclonal antibodies with known binding specificity, such as 9E10, 41C2 and 9B7 can be used to purify viral antigens. These monoclonal antibodies can also be used to enhance antigen recognition by Tn cell clones, facilitating their expansion in the presence of limited amounts of viral antigen.

II. Immunotherapy with T-cell lines

T-cell recognition of different HCMV proteins is important for planning the correct course of immunotherapy with the T-cell lines. The T₂ cells exemplified here primarily recognize structural proteins of HCMV. It is therefore likely that they will be important in the recognition of cell-free viruses (i.e.

in cases of acute viraemia). Data from mice and humans show that certain cytotoxic T cells recognize intermediate-early proteins expressed on the surface of infected cells. As these proteins are only produced after the viral genes are expressed by the host cells, such Tc cells are important to prevent reactivation of latent HCMV, and their delivery is suggested for both preventive and specific HCMV treatment.

The preferred T cell lines recognize viral antigens in association with human LA products. Therefore, therapeutic T cells must be either autologous or share one or more of the restriction specificities expressed by the patient. In the context where isolated T cells are transferred to an individual as a means of treatment, allogeneic cells are cells from a donor individual of the same species as the recipient that are major histocompatibility complex matched for an appropriate human leukocyte antigen class (HLA -class) of antigens expressed by the recipient (abbreviation "MHC LA-matched"). Accordingly, when dealing with a patient who has a pathological infection, such as chronic HCMV disease, one can expand autologous antigen-specific T-cell lines in vitro for subsequent T-cell therapy via re-infusion. Alternatively, when rapid access to T cell clone reagents is important to treat acute, life-threatening infection, pre-expansion and infusion of a number of expanded allogeneic therapeutic T cells of known viral antigen specificity and/or HLA type can be used. This technique is particularly important for patients who

is immunosuppressed.

A further aspect of the present invention is therefore directed to a method for using T-lymphocytes (T-cells) for the therapeutic treatment of a mammal, such as a human patient, who has a viral infection, e.g. HCMV infection. The method comprises treating said infected mammal with an amount of a homogeneous, clonally expanded, viral antigen-specific T-cell population which is effective to produce an increased immune response to the viral infection, said T-cell population being specific (a) for at least one leukocyte antigen (LA) present on the surface of an antigen-presenting cell (APC) of said mammal, and (b) against an antigen of said virus.

Preferably, the clonally expanded T cell population consists mainly of Tn cells or Tc cells. As noted above, it is often preferred to add a number of different (or a "bank") homogeneous clonally expanded virus antigen-specific T cell populations to (a) promote immune system recognition of antigens expressed during different stages of infection and/or (b ) to guarantee that an effective number of the pooled T-cell population will be MHC-matched to the recipient.

The present method for T-cell immunotherapy therefore comprises the supply of a number of different T-cell populations comprising T-cells with specificity (a) for a number of different antigens of said virus, and (b) for at least one LA which present on the surface of an APC of said mammal. Furthermore, the method can also comprise the supply of a number of different T-cell populations, each comprising T-cells with a specificity (a) for at least one antigen of the said virus, and (b) for a number of different LAs present on the surfaces of APCs of a variety of different allotypes of a single species of said mammal, wherein at least one LA is the LA restriction match to said mammal.

Polyclonal lines, as well as T-cell clones, may have significant therapeutic potential. A particular advantage of antigen-specific polyclonal T cell lines is that they include T^ and/or Tc cells that can recognize the target viral antigen in association with all restrictive MHC LAs expressed by the T cell donor. The advantage of virus-specific Tn and Tc clones obtained from a single progenitor T cell is that they represent a homogeneous population of cells with known antigen specificity, functional activity and MHC LA restriction specificity. It is therefore likely that they will show maximum therapeutic effect, as all cells in the population show the same functional activity. From a practical point of view, however, a much larger "bank" obtained from separate (discrete) T-cell populations will be necessary if monoclonal T-cells are to be used.

As the method according to the invention allows the delivery of a therapeutically effective amount of T cells and/or Tc cells, the diseased mammal can be treated, although not necessarily, at the same time with exogenous lymphokines, such as IL-2 (TCGF). As the administration of large doses of IL-2 has been associated with adverse reactions in some patients, the ability to promote the immune response in the absence of IL-2 may provide a significant improvement in the efficacy of T-cell therapy.

Representative antigen-specific T-cell lines which have been generated according to the present methods and which are representative of the populations that can be used to form T-cell "banks" as indicated above, are set forth in Table 2.

The present invention has been described mainly on the basis of the production of Tn and Tc cells which are specific for viral antigens and the treatment of viral infections with homogeneous populations of these T cells. However, it is believed that homogeneous populations of T^ and Tc cells specific for antigens on a variety of different pathological targets can be produced using the present method and that these populations will be effective in enhancing or stimulating the immune response for a variety of different pathogens or pathological targets.

Broadly defined, a pathological target is an entity within the body of a mammal that is the cause of, or the result of, a disease that threatens the health and well-being of the mammalian host. The target may be an entity foreign to the host, such as a virus, bacterium, fungus or the like, or the target may be a product of a disease or pathological condition, such as a virus-infected cell, a neoplastic cell, and the like. In specific embodiments of the therapeutic treatment according to the invention, the pathological target of the treatment is sometimes a secondary infection that is more threatening to the intended recipient, because a primary disease has impaired the recipient's ability to respond immunologically to the secondary infection. Many aspects of primary disease can impair the intended recipient's immune response to the secondary infection, including therapeutic treatments that act to suppress aspects of the recipient's immune system. Such treatments include, but are not limited to, radiotherapy, chemotherapy and the like.

In summary, the present therapeutic method can use allogeneic T cells from individuals of the same species that are MHC LA-matched. This will allow physicians and pharmacists to facilitate the use of preformed dosage forms of allogeneic T cells for the treatment of a patient. Since T cells that are not matched are simply ineffective in helping the patient, the dose may contain many cells, some that are matched and will help the patient and some that are not matched and will not help the patient.

In this way, a single dosage form can be used to treat a number of patients who share one or more LAs expressed by the allogeneic therapeutic cells. Moreover, the dose can contain both Tc and T^ cells which have different MHC class restrictions. It should be obvious that such therapeutic treatment does not require the preparation of doses on a time-to-time basis. This means that the T cells can be expanded in large culture media, allowing the efficiency achieved on a large scale. Moreover, the cells can be prepared so that they are immediately available without requiring individualized collection, leukapheresis and culture.

Regardless of the above advantages, however, the most promising feature of the new immunotherapy is the prospect that each dose will contain antigen-specific T cells. This represents a significant advance over the method according to Rosenberg et al. which only provides non-specific enhancement of cytotoxic cells. In the present method, a dosage form can be provided containing allogeneic T₂ and Tc cells specific for a variety of different antigens all associated with a particular pathogen. E.g. A unit dose can contain a number of

Tn and Tc cell clones that have been individually cultured and then mixed. The dosage form may contain Tn and Tc cells with a preselected number of LA restrictions and also a preselected number of antigen specificities. If a particular pathogen is known to express 10 or 20 known epitopes on a variety of different antigens, structural or otherwise, the dose may contain appropriate T cells that have specificity for each of them. Treatment methodologies will normally involve the parenteral administration of a therapeutically effective amount of autologous or allogeneic T cells in a suitable liquid carrier such as a physiological saline solution. One such method is that provided by S.A. Rosenberg et al., New England J . ' With. , 313, 1485 (1985), but the number of cells delivered per dose and number of doses administered will vary widely, depending on factors determined by the clinician, including the pathology being treated, the patient's physique and physical condition, and other factors. However, due to the improved efficiency of the propagation method according to the invention, it may be possible to remove substantially fewer MNCs from the patient and return substantially more autologous antigen-specific T cells to the patient than is necessary with the methodology according to Rosenberg et al. eel.

Detailed description of the invention

The invention shall be further described in accordance with

the following detailed examples.

EXAMPLE I

1. Production of viral antigens

Human primary fibroblast cultures grown in Dulbecco's modified Eagle<1>s Medium (DMEM) plus 10% fetal bovine serum were infected

with Towne strain HCMV at a multiplicity of infection (MOI) of 1-5. At 3-4 days postinfection, [^H]glucosamine (5 uCi/ml, 22 Ci/mM, Amersham, Arlington Heights, IL) or [-^5S]methionine

(5 uCi/ml, 109 Ci/mM, DuPont/NEN, Boston, MA) added as a marker during subsequent purification for certain of the experiments described below.

Cells and cell debris were removed from the medium by low-speed centrifugation at 7500 x g for 20 min. Viruses were collected from the supernatant by centrifugation at 48,200 x g for 1 hour. The virus pellet was resuspended in Tris NaCl buffer (50

mM trishydroxymethylaminomethane-HCl, pH 7.4, and 150 mM NaCl)

and layered on 20-60% sucrose gradients prepared with the same buffer. Velocity sedimentation was performed at 131,300 x g for 1 hour. Gradients were collected and measured for optical density at 280 nm. A main optical density peak that formed

bands at 43% sucrose were collected. These fractions were diluted with Tris/NaCl buffer and virus collected by centrifugation at 131,300 x g for 2 hours. This purified whole virus preparation was termed "whole HCMV antigen".

To solubilize membrane components, the whole HCMV antigen preparation was resuspended in 2-4 ml of TN buffer (Tris/NaCl buffer (50 mM Tris hydrochloride, pH 7.5, 10 mM NaCl, 2 mM phenyl- methylsulfonyl fluoride)) containing 1% Triton-X 100 (TX-100). After incubation at ambient temperature for 60 minutes,

this solution layered on a linear 20-60% sucrose gradient for rate zonal centrifugation at 120,000 g for 60 minutes. The TX-100 solubilized material remained at the top of the gradient, while the HCMV nucleocapsids were collected in bands by velocity sedimentation. Alternatively, detergent extracts of Towne strain HCMV were prepared using 1.0% Nonidet P-40 (NP-40, Sigma Chemical Company). In this procedure, 10-15 mg of the purified whole HCMV antigen was suspended in 3-5 ml of TN buffer containing 1% NP-40. Detergent/virus suspensions were stirred for 1 hour at room temperature. The extracted proteins were separated from insoluble proteins by high-speed centrifugation for 1 hour. The extract obtained by one or the other of these methods was termed "HCMV detergent extract".

Although HCMV-specific T helper cells appear to recognize HCMV proteins and glycoproteins contained within the whole HCMV antigen preparations, HCMV-specific cytotoxic T cells mainly recognize HCMV-encoded proteins expressed on the surface of the infected cells. Specifically, the major intermediate-early protein (I-E1 protein) expressed on the cell membrane within 6-24 hours of infection appears to be important for the expansion of HCMV-specific cytotoxic T cells.

Autologous fibroblasts or dividing allogeneic fibroblasts

one or more class I leukocyte antigens recognized by the T cell donor were therefore infected with Towne strain HCMV at

a multiplicity of infection of 5-10 for 3 hours. In some cases, the cells were infected in the presence of cyclohexamide (50 µg/ml).

After removal of cyclohexamide, Actinomycin D (5 µg/ml) was added to prevent further DNA transcription, allowing selective expression of I-E genes of HCMV. Infected fibroblasts not treated with cyclohexamide and Actinomycin D expressed both I-E and late gene products of HCMV. These preparations were termed "cell-associated virus antigen".

Herpes simplex virus type I (HSV-I) was partially purified as described by R.C. Gehrz et al., Lancet, 2, 844 (1977) and heat inactivated at 56°C for 1 hour to give "HSV Antigen".

EXAMPLE II

1. Formation of HCMV-specific T-cells and other cytotoxic T-cell lines

HCMV-specific T cell blasts were produced in 25 cm<2> upright tissue culture flasks by stimulation of 10 x 10<6> mononuclear cells (MNC) at a concentration of 1 x 10^ MNC per

ml with 10 µg of heat-inactivated whole HCMV antigen (56°C, 1 hour) suspended in RPMI 1640 medium supplemented with 15% heat-inactivated HCMV-seronegative human serum (PHS). After 7-10 days at

At 37°C in a 5% C02 atmosphere, the HCMV-specific blasts were either expanded further in bulk culture, or cloned by limiting dilution in 9 6-well U-bottomed tissue culture plates with 0.3 cells per well. well in the presence of X-irradiated (5,000 R) autologous MNC (MNC<IRR>) as feeder cells, whole HCMV antigen at a concentration of 1 ug/ml (for Tn cells) or cell-associated antigen with a ratio of fibroblast to MNC of 1:20 (for Tc cells), and 10-20% interleukin-2 (IL-2)

(TCGF, Biotest, Frankfurt, West Germany). The cells were then re-fed every 3-4 days with new IL-2-containing medium. Whole HCMV antigen and autologous x-ray irradiated feeder cells were added to the medium every 7-14 days. day.

Growing cells were fed and transferred to 96-well flat-bottomed tissue culture plates. Growing clones were then transferred to 24-well plates for further expansion and then reseeded in 25 cm<2>tissue culture flasks for large-scale production. Expanded clones were subcloned at another limiting dilution to ensure clonality. Clones were developed from 4 different HCMV-seropositive donors, yielding more than 100 individual T-cell lines. 2. Characterization<g>of HCMV- Tn and TG cell lines and clones All the Tft and Tc lines and clones used in the examples below were characterized with regard to phenotype, proliferation responses, IL-2 production and cytotoxic activity as following:

A. Phenotypic analysis

T cell lines were analyzed for expression of CD phenotypic determinants using the monoclonal antibodies 0KT3 (total T cells), 0KT4 (helper/inducer T cells) and 0KT8 (cytotoxic/suppressor T cells) (Ortho Pharmaceuticals Inc., Raritan, NJ) using an indirect immunofluorescence assay method. Fluorescence was detected either by fluorescence microscopy using a Zeiss fluorescent microscope or flow cytometry using EPICS sp cell 541 (Coulter Corp., Hialeah, FL). Polyclonal T^ lines were predominantly CD3+4+8-; all T^ clones expressed the CD3+4+8 phenotype. The Tc lines were predominantly CD3+4-8+; The Tc clones were CD3+4-8+.

B. Lvmfocvtt propagation

T cell lines and clones were allowed to rest in tissue culture medium in the absence of TCGF overnight and then restimulated for 72 hours with either whole HCMV antigen or HSV antigen to determine the specificity of the proliferative response of T^ clones. All T^ lines and clones showed positive proliferation responses to HCMV, while responses to HSV were the same as for the tissue culture medium background control sample. Thus, all T^ lines and clones were HCMV specific. Tc cell clones proliferated poorly against HCMV antigen in the presence of IL-2.

C. Production of interleukin-2 (IL-2)

Tn clones were stimulated with whole HCMV antigen and the supernatant harvested at 24 h and assayed for IL-2 activity using the mouse cell line CTLL-20 (IL-2 dependent). All Tn clones were shown to produce IL-2, as demonstrated by the survival and growth of CTLL-20. The Tc clones were not tested for IL-2 production.

D. Cvtotoxic activity

All T^ clones were tested for cytotoxic activity against the NK target cell line, K562, autologously uninfected and CMV- and HSV-infected fibroblasts as target cells. No NK or virus-specific cytotoxic activity was observed in any of the T₂ clones.

All Tc clones were tested for cytotoxic activity against the NK target cell lines K5 62, autologous and unmatched or partially matched allogeneic uninfected and HCMV-, HSV- and influenza-infected fibroblasts as target cells. No anti-NK activity was observed with any of the Tc clones. All HCMV-specific Tc clones exhibited cytotoxic activity against autologous and MHC LA-matched HCMV-infected fibroblasts, but not against allogeneic mismatched HCMV-infected fibroblasts. No cytotoxic activity was observed for any of the target cells infected with HSV or influenza virus. Thus, the Tc clones described here exhibit HSMV-specific cytotoxic activity limited by MHC class I antigens and therefore exhibit the characteristics of virus-specific cytotoxic T cells.

EXAMPLE III

Proliferation response of HCMV-specific T^ clones to HCMV antigens

Seventeen HCMV-T^ clones obtained from donor WRC were extensively characterized for phenotype, proliferative responses, IL-2 production and cytotoxic activity. All clones were CD3+4+8-

, proliferative against HCMV but not HSV (as described in Example II (2) above), all produced IL-2 when stimulated with HCMV but not HSV, and none exhibited cytotoxic activity.

Thus, all clones were considered to be T helper cells. All clones also exhibited class II MHC restriction specificity, as determined by blocking with anti-class II monoclonal antibodies and reactivity to whole HCMV antigen presented by autologous or allogeneic antigen-presenting cells sharing the correct Dw restriction determinant expressed in association with DR, DQ or

DP.

For studies of Tc cell reactivity to purified HCMV antigens, X-irradiated autologous mononuclear cells were added as a source of antigen-presenting cells in a 3-day restimulation culture. One uCi per well of tritiated thymidine was added during the last 16 hours of the culture. The cultures were harvested on glass fiber filters and counted on a liquid scintillation spectrophotometer. The results of these analyzes are shown in table 3.

The data set out in Table 3 show that all 17 clones were reactive for the entire Towne-HCMV antigen and showed from 4108 counts per minute to 121,030 counts per minute. These results suggest that the immunodominant determinant(s) recognized by these HCMV-T^ clones are expressed by structural proteins and/or glycoproteins of the virion.

All clones also reacted significantly to Triton X-100 extracts of Towne-HCMV, although to a lesser extent than to whole Towne virus particles. Thus, it seems that most of the Tft clones either primarily recognize proteins or glycoproteins that are included inside the virus envelope or proteins that associate sufficiently with the virus envelope to be recognized in the detergent extract. The lower responses compared to whole Towne antigen may reflect inhibitory effects of residual detergent or a loss of immunogenicity due to structural changes as a result of the extraction procedure.

It is also interesting that all T^ clones also showed a low but significant proliferation response to precipitation of viral antigen after detergent extraction. Although it is likely that some residual envelope glycoproteins and tegument proteins are included within the precipitate, it is also possible that nucleocapsid proteins,

which apparently comprises the dominant protein in the precipitate,

can also express antigenic determinants that are recognized by T-helper cells.

The T cell clones were then stimulated with purified glycoprotein complexes obtained by anion exchange HPLC fractionation of unreduced detergent extract of whole HCMV antigen (see Example I (1) ) •

As stated in B. Kari et al., J. Viroloav. 60, 345 (1986) and in Table 1 above, glycoprotein complexes contained within peaks 2UR and 4UR include a glycoprotein complex recognized by a monoclonal antibody (9E10) This antibody is cross-reactive with various other viruses and is capable of neutralizing HCMV in absence of complement. In contrast, a separate and defined group of glycoprotein complexes is isolated with a high degree of purity in peak 3UR and recognized by a separate class of monoclonal antibodies that react uniquely with HCMV and effectively neutralize HCMV only in the presence of complement.

After the reduction, the most important glycoproteins obtained from the 3UR complex have molecular weights of 130,000, 90,000 and 50,000-52,000 kD, the two glycoproteins obtained from the 2UR/4UR complex recognized by the 9E10 monoclonal antibody have molecular weights of 93,000 and 50,000-52,000 kD. A third glycoprotein has been detected in peak 2UR, which is different from the glycoproteins recognized by the 9E10 monoclonal antibody. Therefore, there are at least three important glycoprotein complexes contained in the HCMV envelope that have been identified as constituting more than 95% of the total glycoproteins within the HCMV viral envelope. As these HPLC methods appear to yield glycoprotein complexes of >95% purity, it was of interest to show the pattern of Tn reactivity towards these individual glycoprotein complexes.

As shown in Table 3, of the 17 T₂ clones tested for reactivity with HPLC-purified glycoprotein complexes, two reacted to peaks 3UR and 4UR, but not to peak 2UR. A third T^ clone reacted to all three HPLC peaks. It is likely that these clones primarily react to the main glycoprotein complex which is also recognized by monoclonal antibodies, which detect the glycoprotein complex mainly isolated in peak 3UR. It is likely that the decreasing part of peak 3UR, as it transitions to peak 4UR, accounts for the apparent cross-reactivity. This specificity was confirmed for Th clones WRC-T3#3, WRC-T2#41 and WRC-L10, which propagated specifically against glycopeptide gpA produced by m-RNA translation of the pgA gene in vitro (data not shown).

Of particular interest is that the majority of clones (14/17) do not appear to recognize determinants expressed by any of the major envelope glycoprotein complexes as represented by materials 2UR, 3UR and 4UR. Thus, the immunodominant HCMV antigenic determinant recognized by these cloned T helper cells may reside either in a non-glycosylated membrane protein or in an internal protein. If this is the case, it may be the same as for the influenza virus, where antibody recognition involves primarily the surface glycoproteins (ie, hemagglutinin), while cytotoxic T lymphocytes are known to recognize primarily internal nucleocapsid proteins.

Table 4 provides data regarding the specificity of HCMV-specific Tft clones reactive with individual HCMV (glyco)proteins. Clones were generated by initial stimulation of MNCs from seropositive donor WRC with whole HCMV antigen to select for Th-reactive with structural proteins and glycoproteins of HCMV.

18% of the Tn clones tested were specific for gpA as detailed in Example 1.1. Representative examples include WRCT3#3 and WRCT2#41. 33% of the HCMV-specific Tn clones were found to be reactive with whole virus as well as a partially purified matrix protein with a molecular weight of 64,000 daltons. This protein was obtained by reverse phase HPLC and gel filtration according to a modification of the method according to B.R. Clark, J. Virol., 49, 279 (1984). Whole Towne-HCMV obtained by centrifugation of the supernatant of the HCMV-infected fibroblasts was solubilized in 6 M guanidinium chloride and run on reverse phase HPLC with a C-18 column. Proteins adsorbed to the column were eluted with acetonitrile and detected by measuring the column outlet current at 214 nm. Peaks collected were pooled, and the 64 kD protein was identified by SDS-PAGE.

The 64 kD protein was further purified from co-eluted proteins by size exclusion chromatography using TSK 4000

and TSK 3000 SEC columns connected in series. An aggregated form of the 64 kD protein was isolated as determined by SDS-PAGE using this method.

Clones WRC-T2/88 and WRC-T2/131 are representative of HCMV-specific T^ clones that proliferated in response to the 64 kD matrix protein. Interestingly, many of the clones (ie, WRC-L33 and WRC-L42) were non-reactive with either the important envelope glycoprotein gpA or the abundant 64 kD internal matrix protein. Thus, it is clear that HCMV-specific T helper cells exist for a number of different structural HCMV proteins.

EXAMPLE IV

Cytotoxic responses of HCMV-specific Tc lines and clones to HCMV antigens

Polyclonal and monoclonal Tc cells were assayed for cytotoxicity against the NK target cell, K562; and autologous or allogeneic fibroblasts infected with HCMV, HSV, or influenza virus. The following specific methodologies were used:

A. Preparation of target cells

1. HCMV-infected human skin fibroblasts (SF)

Human diploid skin fibroblasts (SF) were obtained from core biopsies of adult donors and the foreskin of newborns. Cells were passaged at least 6 times and frozen at a rate of 1°C per minute in Dulbecco's modified Eagle Medium (DMEM) containing 10% fetal calf serum (FCS) and 10% dimethyl sulfoxide (DMSO) and stored in liquid nitrogen. Before use, the cells were thawed in a 37°C water bath, washed with medium and seeded onto 25 cm<2>'s tissue culture flasks. SF were grown to confluence, infected with HCMV at a multiplicity of infection of 0.2 and incubated until 70-90% of the monolayer exhibited cytopathic effect. Cells were then labeled with Na 2 CrC> 4 (375 uCi in 2 ml medium) overnight at 37°C in an atmosphere of 5% CO 2 . The cells were washed three times and resuspended in RPMI in 10% FCS. Uninfected<51>CR-labeled SFs were used as control samples.

2. HSV- and influenza-infected skin fibroblasts

A SF monolayer in a 25 cm<2> flask was infected with HSV or influenza at a multiplicity of infection of 5-10 and incubated overnight. The cells were labeled in the same way as HCMV-infected SF.

3. K562

Erythroleukemia cell line K562 was used as a source of target cells to measure NK activity. K562 cells (1 x 10^) were suspended in 1 ml of medium and 375 uCi of Na2CrC>4 in a 25 cm<21>s flask overnight at 37°C in an atmosphere of 5% CC>2. The cells were resuspended in RPMI supplemented with 10% FCS after three washes.

4. SF that selectively express I-E HCMV proteins

SFer were infected with HCMV at a multiplicity of infection of 5-10 for three hours in the presence of cyclohexamide (15 µg/ml). After removal of cyclohexamide, Actinomycin-D (5 µg/ml) and Na 2 CrO 4 (375 µCi) were added to the culture. The cells were then washed three times and resuspended in RPMI in 10% FCS as target cells. The cytotoxic measurement was performed under Actinomycin-D to prevent DNA transcription. Uninfected SFs treated with cyclohexamide and Actinomycin-D in parallel were used as comparison samples.

B. Cvtotoxicity measurement

Three thousand target cells in 100 µl were added to each well of V-bottomed microtiter plates. Serial dilutions of effector cells in 100 µl were then added to the target cells to give a ratio of effector cells to target cells of 50:1, 25:1 and 12:1. The plates were centrifuged at 50 g for 5 minutes at 37°C in an atmosphere of 5% CO 2 for 10 hours. 100 µl supernatants were harvested and counted in a gamma counter (Beckmann Gamma 9000). The comparison samples consisted of maximum lysis and spontaneous release which was achieved by adding 100 µl of Triton-X-100 in medium or target cells. Calculations are carried out according to the following formula:

Experimental counts per minute represented cells from wells with effector cells.

The results were expressed as the mean value of wells in triplicate or quadruplicate. Spontaneous release from both infected and uninfected SF target cells was less than 40%.

1. Cytotoxic activity of HCMV-specific pol<y>clonal Tc cell lines stimulated with Towne HCMV-infected autologous

skin fibroblasts (SF)

Mononuclear cells from seropositive donor SP-CN were stimulated for 7-10 days in mass culture with autologous skin fibroblasts (SF) infected for 20 hours with Towne HCMV. T-cell blasts were restimulated weekly with HCMV-infected fibroblasts, autologous irradiated MNC as feeder cells, and IL-2. Resulting polyclonal CD8+ T cell lines were evaluated for cytotoxic activity against uninfected and HCMV-infected autologous fibroblasts and the NK target cell line K562. The results of this survey are shown in table 5.

As can be seen from the data presented in Table 5, both cell lines exhibited cytolytic activity against autologous fibroblasts infected for 20 hours with HCMV, expressing intermediate-early and late viral proteins and against CMV-infected autologous fibroblasts that had been treated with cyclohexamide (CH) and Actinomycin-D (act. D) for selective expression of the intermediate-early proteins of HCMV. Both lines also express significant levels of NK-like cytotoxic activity.

2. Cytotoxic activity of HCMV-specific Tc cell lines, including clones stimulated with sucrose radient/purified Towne HCMV virus particles

CD8+ Tc cell lines and clones were obtained from seropositive donor SP-RK by initial stimulation of MNC with sucrose gradient-purified Towne-HCMV virus particles, followed by repeated stimulation with HCMV virus particles, autologously irradiated MNC as feeder cells and IL-2. Three CD8+ lines and clone CD8+ SP-RK #3 lysed HCMV-infected autologous fibroblasts, but not allogeneic mismatched HCMV-infected fibroblasts or autologous fibroblasts infected with either HSV or influenza virus as shown in Table 6.

These lines expressed little cytotoxic activity against K562-NK target cells. Thus, these T-cell lines and clones are characteristic of HCMV-specific MNC class-I LA-restricted T-cytotoxic cells that recognize one or more antigens that express whole virus antigens (ie, structural proteins.

EXAMPLE V

Enhancement of antigen-induced proliferation of HCMV-specific Tn clones using polyclonal antiserum from seropositive donors and HCMV-specific monoclonal antibodies

1. Monoclonal antibody

HCMV-Th clone WRC-T3#3 was used at a concentration of 1.5 x 10<4> cells per well as a source of responder cells. Autologous X-irradiated MNC (10^) were added as a source of antigen presenting cells. An optimal dilution of whole HCMV antigen or 0.1 µg/ml of HPLC-purified 3UR was added as a source of HCMV-specific antigen stimulation. Serial dilutions of monoclonal antibody 41C2, which recognizes the immunodominant glycoprotein A (gpA) or a control monoclonal antibody 35F10, which recognizes a non-glycosylated protein designated protein D were added at concentrations ranging from 10 down to 0.01 µg/ ml of protein per well. The results of this experiment are set out in Table 7.

The data presented in Table 7 show that monoclonal antibody 41C2 significantly enhanced the proliferative response of the Tn clone when 3UR or whole HCMV antigen was used for antigenic stimulation. In contrast, no significant enhancement was observed with the monoclonal antibody directed against protein D using either 3UR or whole HCMV antigen.

Thus, it appears that monoclonal antibodies directed against an immunogenic glycoprotein complex that specifically stimulates a particular Tft clone are effective in enhancing the proliferative response.

Six HCMV-T^ clones shown to respond to whole HCMV antigen were then stimulated with whole HCMV antigen plus serial dilutions of either monoclonal antibody 41C2 (to gpA) or monoclonal antibody 35F10 (to protein D). Seropositive and seronegative whole sera were also tested under the same conditions. The results of these experiments are shown in Table 8.

It was surprising that monoclonal antibody 41C2, directed against gpA, enhanced the proliferative response of all six T^ clones to whole HCMV antigen despite the fact that only 3 of the 6 clones reacted to HPLC-purified gpA in the absence of antibody (see table 7). The monoclonal antibody directed against protein D did not enhance the response of any of the clones tested. Whole serum from the seropositive donor enhanced the response of all clones, whereas that from the seronegative donor did not. These data suggest that the enhancement mechanism does not necessarily involve a direct interaction between the antibody and the specific viral protein or glycoprotein recognized by the T₂ clone.

EXAMPLE VI

Presentation of HCMV antigen to T helper cell clones by MNC,

LCL or mixtures thereof

1.Epstein-Barr virus-transformed (EBV-transformed) lvmfo-blastoid cell lines (LCL)

An autologous LCL from seropositive donor SP-CN (SP-CN LCL) was created by the method of Sugden and Mark, J. Virology, 23, 503 (1977) and cultured in RPMI 1640 medium (GIBCO, Grand Island, NY), supplemented with 10% heat-inactivated fetal calf serum (FCS). AMG LCL (DR 2.2/D2 2.2) and NHS LCL (DR 3.8) were gifts from Dr. F. Bach {IRC, University of Minnesota, Minneapolis, MN).

2. Generation of HCMV-specific T cell clones

HCMV-specific T-cell clones were generated from a seropositive donor (SP-CN). Briefly, mononuclear cells (MNC) were cultured at 10^ cells per ml in RPMI 1640 medium supplemented with 15% heat-inactivated HCMV seronegative human serum (PHS) and 1 µg/ml whole HCMV antigen. After 7-10 days, T-cell blasts were isolated and cloned by limiting dilution at 0.3 cells per well in round-bottomed wells containing 0.2 ml RPMI/15% PHS medium with 10,000 autologous X-irradiated (5000 R) MNC, 1 µg/ml whole HCMV antigen (Towne strain) and 15% IL-2 (TCGF, Biotest, Frankfurt, West Germany). Plating efficiencies of 2-12% were observed. The clones were expanded in culture medium containing 10-20% IL-2 and restimulated with autologous X-rayed MNC/LCL mixture and 1 µg/ml whole HCMV antigen every 1-2 weeks.

3. Formerinasanalvse

HCMV-specific cloned Tn cells were analyzed 7-14 days after restimulation. The microcultures were arranged in round-bottom plates (Flow Lab., Inc., VA) with a total volume of 0.2 ml per well containing 10<4>HCMV-specific T^ cells, various concentrations of X-irradiated analog MNC and/or LCL and whole HCMV antigen (Towne strain) or HSV antigen. The cultures were incubated for 3 days, and 1 uCi per well<3>H-thymidine (New England Nuclear, Boston, MA) was added to each well for the last 16-24 hours of cultivation. The wells were harvested with an automatic cell harvester, the cells collected on glass fiber filter paper and radioactivity measured in a Beckman 5801 liquid scintillation spectrometer.

4.LCL presents HCMV antigen to HCMV-specific T^ clones A panel of HCMV-specific T^ clones generated from a seropositive donor (SP-CN) was tested for HCMV-specific proliferative activity using either X-irradiated autologous MNC( IO<5>per well) or x-rayed autologous LCL (10<4>- 3 x 10<4>per well) as APC. HSV antigen was included as an antigen-specific control sample. Table 9 shows the results obtained using seven clones.

All of the T₂ clones listed in Table 9 proliferated well in response to HCMV antigen but not HSV antigen when MNC were added to the cultures as APC. The degree of multiplication varied from 8,114 to 68,307 cmp. Most of these clones also showed HCMV antigen-specific proliferative responses when LCL was used as APC with a range from 1,020 to 21,370. Clones SP-CN/T2-131 and SP-CN/T5-43 proliferated well against HCMV in the presence of irradiated autologous MNC, but poorly in the presence of

autologous LCL.

On the other hand, clones SP-CN/T3-16 and SP-CN/T3-9 proliferated well against HCMV in the presence of irradiated autologous LCL, although their proliferation against HCMV in the presence of cultured MNC was no better than that of the others five clones. Thus, there is no apparent correlation between the proliferation response to HCMV using MNC as APC and that obtained using LCL as APC. It is well known that antigen-specific T helper cells (T- cells) recognize antigen associated with class II MHC products on the surface of APC-(P. Erb et al., J. Exper.

Med., 142, 460 (1975)). The inventors examined the MHC restriction of some of these T₂ clones using LCL as APC and found that all clones tested are class II restricted. LCLs from other MHC class II-matched donors also presented HCMV antigen well to the two high-reacting clones (SP-CN/T3-16 and SP-CN/T3-9), but poorly to the five low-reacting clones. E.g. presented CP-CN LCL (autologous, DR2, 4/DW/2,4) as well as AMG LCL (DR2,2/DW2,2) HCMV antigen against clone SP-CN/T3-9, while NHB LCL (DR3,8 ) did not present HCMV for the same clone.

5. Enhancing effect of LCL on proliferative responses of T cells to MNC and HCMV

Due to limited supplies of autologous MNC as APC to stimulate HCMV-specific T^ cells and ineffective antigen presentation by autologous LCL when used alone, combinations of MNC and LCL were used as APC and feeder cells during reactivation of T^- the clones. The combination of LCL and MNC surprisingly showed a synergistic effect on proliferative response, as shown in Table 10.

LCL alone as APC in an area of 10<4->10<5>LCL per well did not stimulate any significant T-cell proliferative response to whole HCMV antigen above background. The addition of LCL at 10<4>LCL per well and 3 x 10<4>LCLpr. well, however, significantly enhanced the 3-day proliferation response of T^ cells to MNC plus whole HCMV, although IO<5>LCL per well appeared to suppress the proliferation responses, probably due to excessive cell density in the culture.

The same cultures were expanded in the presence of 15% IL-2 for 12 days, and cell counts were performed on day 7 and day 12 after initiation of culture. The results of these experiments are set forth in Table 11. As shown in Table 11, when no MNC was present, Tn cells were not expanded even when both LCL and whole HCMV antigen were added to the culture. A combination of MNC and whole HCMV antigen led to a 3-43 fold increase in T-cell numbers over 12 days of culture depending on the amount of MNC involved. When LCL was added together with MNC and HCMV antigen, a 29-620-fold increase in cell number was observed over the same time period under the same tissue culture conditions, clearly indicating that LCL enhanced the expansion of activated Tn cells.

Portions of these cultures were plated in 96-well flat-bottomed microtiter plates on day 7 at 0.2 ml per well. well in triplicate. After overnight pulsing with [^Hj-thymidine, a similar effect of LCL on cell proliferation was observed as measured by incorporated [%]-thymidine as indicated in Table 12.

These results confirmed the feasibility of using both MNC and LCL as feeder cells together with antigen in the activation and expansion of T^ clones. The extent of T^ cell expansion is proportional to the concentration of MNC<IRR> in the range from 10<4->10<5>MNC per well, but not proportional to the concentration of LCL<IRR>. The optimal ratios between LCL and cloned T₂ cells range from 1:1 to 3:1.

Samples of antigen-presenting LCL cell lines SP-CN/T3-43 (accession code: IVI-10122), and SP-CN/T5-43 (accession code: IVI-10123)

has also been deposited with In Vitro International, Linthicum, MD,

according to the Draft PTO Deposit Policy for Biological Materials, BNA PTCJ, 32, 90 (1986).

Cultures of these deposited cell lines will be made available to the public by issuing a patent based on the present application. It is to be understood that the availability of the deposit does not constitute a license to practice the subject invention as a waiver of the patent rights granted by the United States Government. Although certain representations of embodiments are described herein for the purpose of illustrating the invention, it should be understood by those skilled in the art that modifications may be made without departing from the scope and spirit of the invention.

Thanks are extended to Children's Hospital, St. Paul, Minnesota, for generous support and assistance in this work.

Claims (48)

1. Process for producing a homogeneous population of T-helper cells that are specific for a viral antigen, characterized in that it comprises the following steps: (a) isolating a population of mononuclear cells (MNC) from the blood of a donor mammal, wherein the MNC population comprises T helper cells specific for said viral antigen; and autologous, non-proliferative antigen-presenting cells (APC); (b) combining said isolated population of said MNCs with an amount of said viral antigen effective to cause the proliferation of a T helper cell specific for said antigen; (c) allowing said T helper cell to proliferate for a period of time effective to allow non-proliferating MNCs to lose viability; and (d) clonally expanding said antigen-specific T helper cell in the presence of a population of non-proliferating antigen-presenting cells comprising a mixture of (i) autologous MNCs, allogeneic MNCs or mixtures thereof, and (ii) autologous lymphoblastoid cells (LCLs ), allogeneic LCLs or mixtures thereof and an amount of the said viral antigen which is effective for the propagation of the said cloned antigen-specific T-helper cell, to give the said homogenic population, and where the said LCLs are present in an amount which is effective to cooperate with the MNCs to increase the proliferation rate of the T-cell. 2. A method as set forth in claim 1, characterized in that additional amounts of said viral antigen and said autologous, non-proliferating APCs effective to cause the proliferation of a T helper cell are added during step (c) to cause further multiplication of said T-helper cell. 3. Method as stated in claim 1, characterized in that in step (d) the non-proliferating APCs comprise a mixture of (i) autologous MNCs, and (ii) autologous LCLs, which are effective for propagating the said antigen-specific T- helper cell, in that the aforementioned LCLs are present in an amount which is effective for cooperating with the MNCs to increase the multiplication rate of the T helper cell. 4. Method as stated in claim 1, characterized in that the LCLs are obtained from a virus-transformed lymphoblastoid cell line. 5. Method as stated in claim 4, characterized in that the virus-transformed lymphoblastoid cell line is an Epstein-Barr virus-transformed (EBV-transformed) lymphoblastoid cell line. 6. Method as stated in claim 1, characterized in that said virus antigen is a human cytomegalovirus antigen (HCMV antigen). 7. Method as stated in claim 6, characterized in that said HCMV antigen is a viral structural protein. 8. Method as stated in claim 7, characterized in that the HCMV antigen is present in a virus envelope fraction. 9. Method as stated in claim 7, characterized in that the HCMV antigen comprises a glycopeptide complex that is present on the virus surface or a viral glycopeptide that is obtained therefrom. 10. Method as stated in claim 9, characterized in that the HCMV antigen is an immunogenic glycopeptide of approximately 93 kD or an immunogenic glycopeptide of 50-52 kD, which glycopeptides are present on the virus surface. 11. Method as stated in claim 6, characterized in that the HCMV antigen is an approximately 64 kD matrix protein. 12. Method as set forth in claim 1, characterized in that step (b), (c) or (d) is carried out in the presence of an amount of monoclonal antibody which is effective for increasing the multiplication rate of said T-helper cell, the monoclonal antibody is specific for said viral antigen. 13. Method as stated in claim 12, characterized in that the monoclonal antibody is specific for human cytomegalovirus antigen (HCMV antigen). 14. Method as stated in claim 13, characterized in that the monoclonal antibody is specific for an immunogenic glycopeptide with a molecular weight of 50-52 kD or an immunogenic glycopeptide with a molecular weight of approximately 93 kD which is present on the virus surface. 15. Method as stated in claim 14, characterized in that the monoclonal antibody is produced by hybridoma IVI-10117, ivi-10118 or IVI-10119. 16. Method as stated in claim 1, characterized in that step (b), (c) or (d) is carried out in the presence of an effective amount of interleukin-2 (IL-2). 17. Method for producing a homogeneous population of T-helper cells that are specific for a viral antigen, characterized in that it comprises the following steps: (a) isolating a population of mononuclear cells (MNC) from the blood of a donor mammal, the MNC population comprising T helper cells specific for said viral antigen and autologous, non-proliferating antigen-presenting cells (APC); (b) combining said isolated population of said MNCs with an amount of said viral antigen and an amount of a monoclonal antibody specific for said viral antigen effective to cause the proliferation of a T helper cell specific for said antigen; (c) allowing said T helper cell to proliferate for a period of time effective to allow non-proliferating MNCs to lose viability; and (d) clonally expanding said antigen-specific T helper cell in the presence of a plurality of non-autologous antigen-presenting cells or mixtures thereof; an amount of said viral antigen, and an amount of said monoclonal antibody specific for said antigen, which amounts are effective for propagation of said cloned antigen-specific T helper cell, to give said homogeneous population. 18. Method as stated in claim 17, characterized in that the viral antigen is an HCMV antigen. 19. Method as stated in claim 17, characterized in that in step (d) the autologous non-proliferating APCs comprise non-proliferating MNCs. 20. Method as stated in claim 19, characterized in that in step (d) the autologous APCs or the allogeneic APCs further comprise non-proliferating LCLs in an amount which is effective to further increase the rate of multiplication of the T-helper cell in relation to the increase which is caused by the non-propagating MNCs. 21. Process for producing a homogeneous population of T-helper cells that are specific for an antigen present on a human cytomegalovirus (HCMV) structural protein, characterized in that it comprises the following steps: (a) isolating a population of mononuclear cells (MNC) from the blood of a donor mammal, wherein the MNC population comprises T helper cells having specificity for said human cytomegalovirus antigen (HCMV antigen), and non-proliferating antigen presenting cells (APC); (b) combining said isolated MNC population with an amount of said human cytomegalovirus antigen (HCMV antigen) effective to cause the proliferation of a T helper cell specific for said human cytomegalovirus antigen (HCMV -antigen); (c) allowing said T helper cell to proliferate for a period of time effective to allow non-proliferating MNCs to lose viability; and (d) clonally expanding said antigen-specific T helper cell in the presence of an effective amount of said HCMV antigen, a monoclonal antibody specific for said human cytomegalovirus antigen (HCMV antigen), and an autologous antigen-presenting cell selected from the group of x-irradiated autologous MNCs, an x-irradiated Epstein-Barr virus-transformed (EBV) lymphoblastoid cell line (LCL) and mixtures thereof, to provide said homogeneous population. 22. Method as set forth in claim 21, characterized in that additional amounts of said viral antigen, said monoclonal antibody specific for said viral antigen and said autologous, non-proliferating APCs, which are effective to cause the propagation of said T-helper cell, is added during step (c) to cause further proliferation of said T-helper cell. 23. Method as stated in claim 21, characterized in that step (b), (c) or (d) is carried out in the presence of an effective amount of interleukin-2 (IL-2). 24. Method for propagating a homogeneous population of T-helper cells specific for a viral antigen, comprising combining said homogeneous population of T-helper cells with an amount of said viral antigen and an amount of a mixture of non-proliferating, antigen-presenting cells (APC) operative to cause the proliferation of said population of T helper cells, said mixture of APCs comprising (i) autologous or allogeneic virus-transformed lymphoblastoid cells (LCL) and (ii) autologous or allogeneic mononuclear cells ( MNC), in that the LCLs are present in an amount that is effective to cooperate with the MNCs to increase the multiplication rate of the T-helper cells. 25. Method as stated in claim 24, characterized in that the ratio between LCLs and MNCs is at least 1:1-1:10. 26. Method as stated in claim 25, characterized in that said virus antigen is a human cytomegalovirus antigen (HCMV antigen). 27. Method as set forth in claim 24, characterized in that it further comprises combining said population of T-helper cells with an amount of a monoclonal antibody that is specific for said antigen, which is effective to increase the multiplication rate of said population of T helper cells. 28. Procedure as specified in claim 24: characterized in that it further comprises combining the said population of T-helper cells with an amount of interleukin-2 (IL-2) which is effective to increase the multiplication rate of the said population of T-helper cells. 29. Method as stated in claim 24, characterized in that the ratio between T-helper cells and LCLs is 1:1-3:1. 30. Method for producing a homogeneous population of cytotoxic T cells specific for a human cytomegalovirus antigen (HCMV antigen), characterized in that it comprises the following steps: (a) isolating a population of mononuclear cells (MNC) from the blood of a donor animal, wherein the MNC population comprises cytotoxic T cells specific for HCMV virus antigens; (b) combining said isolated population of said MNCs with (i) a population of autologous, non-proliferating, antigen-presenting cells (APCs), (ii) a population of HCMV-infected autologous fibroblasts or whole HCMV antigen, and (iii) an amount of interleukin-2 (IL-2) effective to cause the proliferation of a cytotoxic T cell specific for an HCMV antigen expressed by said APCs; (c) allowing said T helper cell to proliferate for a period of time effective to allow non-proliferating MNCs to lose viability; and (d) clonally expanding said antigen-specific cytotoxic T cell in the presence of (i) a plurality of HCMV-infected, non-proliferating, autologous antigen-presenting cells, HCMV-infected non-proliferating, allogeneic antigen-presenting cells, or mixtures thereof, ( ii) an amount of IL-2 and (iii) an amount of autologous HCMV-infected fibroblasts or whole HCMV antigen effective for propagation of said cloned antigen-specific cytotoxic T cell, to provide said homogeneous population. 31. Method as stated in claim 30, characterized in that said donor mammal is a human. 32. Method as stated in claim 30, characterized in that further amounts of said viral antigen, said IL-2 and said autologous, non-proliferating APCs which are effective to cause the proliferation of said cytotoxic T-cell are added during of step (c) to cause further proliferation of said cytotoxic T cell. 33. Method as stated in claim 31, characterized in that in step (b) the population of MNCs is combined with HCMV-infected autologous fibroblasts. 34. Method as set forth in claim 33, characterized in that in step (d) said antigen-presenting cells further comprise non-proliferating MNCs obtained from a continuous autologous MNC line in an amount effective to increase the rate of proliferation of the cytotoxic T -cells in cooperation with the autologous mononuclear antigen-presenting cells. 35. Method as stated in claim 34, characterized in that the MNC line comprises a virus-transformed lymphoblastoid cell line. 36. Method as stated in claim 35, characterized in that the virus-transformed lymphoblastoid cell line comprises an Epstein-Barr virus-transformed (EBV-transformed cell line). 37. Method as stated in claim 30, characterized in that step (b), (c) or (d) is carried out in the presence of an amount of a monoclonal antibody which is effective to increase the rate of multiplication of said cytotoxic T-cell, wherein the monoclonal antibody is specific for an HCMV antigen. 38. Method for propagation of a homogeneous population of cytotoxic T cells specific for an antigen associated with a pathogen, characterized in that it comprises combining said homogeneous population of cytotoxic T cells with an amount of interleukin-2 (IL-2) and an amount of a mixture of non-proliferating, antigen-presenting cells (APC) effective to cause the propagation of said population of cytotoxic T cells, said mixture of APCs comprising allogeneic virus-transformed lymphoblastoid cells (LCL) and allogeneic mononuclear cells (MNC) expressing said antigen. 39. Method as stated in claim 38, characterized in that said antigen is a viral antigen. 40. Method as stated in claim 39, characterized in that said virus antigen is a human cytomegalovirus antigen (HCMV antigen). 41. Method as set forth in claim 38, characterized in that it further comprises combining the said population of cytotoxic T cells with an amount of monoclonal antibody that is specific for the said antigen, which is effective for increasing the multiplication rate of the said cell population . 42. Pharmaceutical unit dosage form, characterized in that it comprises a quantity of a homogeneous population of antigen-specific T-lymphocytes comprising Tn cells or Tc cells which are effective to increase an immune response in a recipient mammal for a pathological target antigen upon parenteral administration of said dosage form, wherein said population of T-lymphocytes is MHC LA-matched with respect to the recipient mammal, and wherein said T-lymphocytes are antigen-specific for said pathological target antigen. 43. Pharmaceutical unit dosage form, characterized in that it comprises a mixture of a number of homogeneous populations of antigen-specific T lymphocytes, where said mixture comprises T^ cells, Tc cells or mixtures thereof, each of said populations being specific for a pathological target antigen, where at least one of said plurality of T-lymphocyte populations is MHC LA-matched with respect to a recipient mammal, and wherein said composition is effective to increase an immune response in the recipient mammal to at least one pathological target antigen upon parenteral administration of said dosage form . 44. Pharmaceutical unit dosage form, characterized in that it comprises a number of separately packaged homogeneous populations of antigen-specific T-lymphocytes comprising Tn cells or Tc cells, each population comprising T-lymphocytes that are specific for a pathological target antigen, each population being active to increase an immune response in an MHC-matched recipient mammal for said pathological target antigen by parenteral administration of said population of T-lymphocytes, and wherein at least one of said populations is MHC LA-matched with respect to the recipient mammal. 45. Pharmaceutical unit dosage form, characterized in that it comprises a quantity of a heterogeneous population of antigen-specific T lymphocytes comprising Th cells, Tc cells or mixtures thereof for effectively increasing an immune response in a recipient mammal against at least one pathological target antigen by parenteral administration of the said population of T lymphocytes, and wherein at least a portion of said population of T lymphocytes is MHC LA matched with respect to the recipient mammal. 46. Pharmaceutical unit dosage form, characterized in that it is produced by a method which comprises combining a number of homogeneous populations of antigen-specific T-lymphocytes comprising T^ cells or Tc cells with a pharmaceutically acceptable liquid carrier, where each T-lymphocyte population is antigen-specific for a pathological target antigen, and wherein at least one T lymphocyte population is MHC LA matched with respect to the mammalian recipient. 47. Pharmaceutical unit dosage form as stated in one of claims 44-46, characterized in that said a number of T-lymphocyte populations comprise antigen specificities for a number of antigens which are associated with a particular pathological target. 48. Pharmaceutical unit dosage form as stated in one of claims 44-46, characterized in that said a number of T-lymphocyte populations comprise antigen specificities for a number of HCMV-associated antigens.