WO1995007105A1

WO1995007105A1 - Haplotype-matched cytokine-secreting cells and methods of using to stimulate an immune response

Info

Publication number: WO1995007105A1
Application number: PCT/US1994/010217
Authority: WO
Inventors: Robert E. Sobol; Ivor Royston; Habib Fakhrai
Original assignee: Sidney Kimmel Cancer Center
Priority date: 1993-09-07
Filing date: 1994-09-06
Publication date: 1995-03-16
Also published as: CA2171211A1; EP0721351A1; EP0721351A4; JPH09504786A

Abstract

The invention relates to a composition comprising haplotype-matched tumor cells which have been genetically modified to express a cytokine gene and to a composition comprising autologous fibroblasts which have been genetically modified to express a cytokine gene and unmodified tumor cells. The invention also relates to methods for using the haplotype-matched cytokine-secreting cells to stimulate an immune response against a tumor located in the central nervous system of a cancer patient. The invention further relates to a method of inhibiting or preventing the growth of tumor cells in the central nervous system of a patient by immunizing the patient with the haplotype-matched cytokine-secreting cells.

Description

HAPLOTYPE-MATCHED CYTOKINE-SECRETING CELLS AND METHODS OF USING TO

STIMULATE AN IMMUNE RESPONSE

BACKGROUND OF THE INVENTION

FIELD OF THE INVENTION

This invention relates generally to the fields of gene therapy and i munotherapy of cancer and, more specifically, to a composition comprising haplotype-matched tumor cells which have been genetically modified to express a cytokine gene and to a composition comprising autologous fibroblasts which have been genetically modified to express a cytokine gene and unmodified tumor cells. The invention also relates to methods for using the haplotype-matched cytokine-secreting cells to stimulate an immune response against a tumor located in the central nervous system of a cancer patient. The invention further relates to a method of inhibiting or preventing' the growth of tumor cells in the central nervous system of a patient by immunizing the patient with the haplotype-matched cytokine-secreting cells.

BACKGROUND INFORMATION

Cytokines are immune system modulators that mediate many of the immune responses involved in anti-tumor immunity. Several cytokines have been produced using recombinant DNA methodology and evaluated for their effectiveness in treating cancer patients. The administration of lymphokines, which are cytokines produced by lymphocytes, and related im unomodulators has produced positive responses in patients with various types of neoplasms. However, administration of cytokines frequently is associated with toxicity, which limits the therapeutic value of these agents. Interleukin-2 (IL-2) is a lymphokine having a central role in the generation of anti-tumor immunity (Rosenberg et al., J. Natl. Cane. Inst., 10:73-77 (1990)). In response to tumor antigens, helper T cells secrete small quantities of IL-2, which acts locally at the site of a tumor antigen to activate cytotoxic T cells and natural killer cells. The latter cells effect systemic tumor cell destruction.

Intravenous, intralymphatic and intralesional administration of high doses of IL-2 have produced clinically significant responses in some cancer patients.

However, severe toxicity, including hypotension and edema, limit the dose and, therefore, the usefulness of intravenous and intralymphatic IL-2 administration (see Lotze et al, J. Amer. Med. Assn.. 256:3117-3124 (1986);

Pizza et al., Lymphokine Res. , 7:45-48 (1988); Gandolfi et al., Hepato-Gastroenteroloqy, 36:352-356 (1989); Sarna et al., J. Biol. Resp. Mod., 9:81-86 (1990)). The toxicity of systemically administered lymphokines is not surprising as these agents mediate local cellular interactions and normally are secreted in very small quantities.

Other cytokines, such as interleukin-4 , interleukin-7, colony stimulating factors, alpha-interferon and gamma-interferon also have been used to stimulate immune responses to tumor cells (see, for example, Hock et al., J. Exp. Med.. 174:1291-1298 (1991); Thomassen et al., Cane. Res.. 51:857-862 (1991), each of which is incorporated herein by reference). Like IL-2, however, current modes of administration of these cytokines cause adverse side effects to the patient.

To circumvent the toxicity of systemic cytokine administration, the effectiveness of intralesional injection of IL-2 has been investigated. While this approach eliminates the toxicity associated with systemic IL-2 administration, multiple intralesional injections are required to optimize therapeutic efficacy (Bubenik et al., Immunol. Lett. , 19:279-282 (1988); Bubenik et al., Immunol. Lett. , 23:287-292 (1989/1990)). Thus, these injections are impractical for many patients, particularly when tumor sites are inaccessible for injection or create a risk of patient morbidity.

An alternative approach to cytokine therapy involves inserting cytokine genes into tumor cells. Using this method, the expression of cytokine gene products following cytokine gene transfer into the tumor cells abrogates the tumorigenicity of the cytokine-secreting tumor cells when implanted into syngeneic hosts. The transfer of genes encoding IL-2 (Fearon et al., Cell, 387- 403 (1990); Gansbacher et al., J. EXP. Med.. 172:1217-1224 (1990)), gamma-interferon (Watanabe et al., Proc. Natl. Acad. Sci.. USA. 86:9456-9460 (1989)), interleukin-4 (Tepper et al., Cell. 57:503-512 (1989)) or granulocyte- macrophage colony stimulating factor (Dranoff et al., Proc. Natl. Acad. Sci. USA . 90:3539-3543 (1993)) significantly reduces or eliminates the growth of several different histological types of urine tumors.

In the studies employing tumor cells genetically modified to express IL-2, treated animals rejected the cytokine-secreting tumor cells and developed systemic immunity against the tumor cells and were protected against subsequent tumor cell challenge with unmodified tumor cells (see Fearon et al., 1990, and Gansbacher et al., 1990). Similar inhibition of tumor growth and protective immunity also was demonstrated when immunizations were performed with a mixture of unmodified parental tumor cells and genetically modified tumor cells, which expressed IL-2. No toxicity associated with localized lymphokine transgene expression was reported in these animal tumor studies (see Fearon et al., 1990; Gansbacher et al., 1990; and Tepper et al., 1989; see, also, Kriegler et al., Gene Transfer and Expression: A laboratory manual (Stockton Press 1990), which is incorporated herein by reference) .

It was estimated that 15,600 new cases of primary brain and CNS tumors would occur in 1990 (Ransohoff et al., Cancer of the Central Nervous System and the Pituitary, in American Cancer Society Textbook of Clinical Oncology, Holleb et al., ed. , Chap. 24 (American Cancer Society, Inc. 1991)). In addition, many non-CNS primary tumors metastasize to the CNS. As the lifespan of humans increases, the number of cancer patients having CNS involvement is expected to increase.

The prognosis for brain cancer patients is poor. For example, of the estimated 12,000 new cases of primary CNS tumors in 1987, 11,100 deaths were projected in 1990 (Ransohoff et al. 1991). Furthermore, all patients with primary CNS tumors of the glioma series will eventually die of their disease. Although there is no clear line of demarcation, tumors of the glioma series are generally classified as benign astrocytomas, anaplastic astrocytomas or glioblastoma (referred to herein generally as "gliomas") (Ransohoff et al. 1991).

For example, gliomas currently are treated by surgery, radiotherapy or chemotherapy, either alone or in various combinations (Levin et al., Int. J. Rad. Oncol. Biol. Phvs.. 18:321-324 (1990)). In addition, investigational treatments have been utilized, including local administration of IL-2 in combination with autologous lymphokine activated killer cells (Ransohoff et al. 1991). However, the effectiveness of this method has not been established and, in any case, suffers from the morbidity problems associated with localized injection of such a formulation at the site of the tumor as discussed above. In view of the problems associated with localized treatment in the CNS using, for example, cytokine-secreting tumor cells or lymphocyte activated killer cells, it would be advantageous if an active immune response could be induced in a site other than the CNS, yet still provide an effect at the site of the tumor. However, it is unclear whether the induction of an active immune response outside the CNS will provide a therapeutic advantage against a tumor within the CNS. For example, the CNS is considered to be a partially immunologically privileged site (Oldfield et al.. Hum. Gene Ther. , 4:39-69 (1993)), suggesting that immune effector cells are not active across the blood-brain barrier. This "immunologic privilege" makes it merely speculative that immunization with genetically modified tumor cells will induce an immune response against the tumor cells within the CNS. In fact, a recent report by Ram et al. (Abstract, presented at the Meeting of the Amer. Assn. Neurosurg., Boston, MA, 1993) indicates that an immune response against tumor cells that were genetically modified to express the IL-2 gene does not occur in the CNS. Using the rat brain tumor model, 9L, Ram et al. genetically modified the 9L tumor cells by introducing a gene encoding IL-2. When the IL-2-secreting tumor cells were injected peripherally, the majority of animals did not form tumors. However, when the IL-2-secreting 9L cells were injected in the CNS, tumors were observed to grow in the rat CNS. These results in the CNS are in contrast to the results reported in other tumor systems using tumor cells genetically modified to express IL-2 (see, for example, Fearon et al., 1990, and Gansbacher et al., 1990). These results indicate that the CNS is an immunologically privileged site and that results using cytokine gene therapy in other tumor systems are not predictive of the therapeutic value of such therapy in treating CNS tumors.

Mahaley et al., J. Neurosurg. , 59:201-207 (1983), immunized glioma patients with subcutaneous injections of allogeneic human glioma tissue culture cell lines. The patients also were treated with levamisole and with radiotherapy and BCNU chemotherapy. Patients inoculated with the U-251MG cell line had significantly longer survival compared to non-immunized historical control patients treated with levamisole, radiotherapy and chemotherapy. However, while the results of Mahaley et al. appeared promising, more recent studies using similar methods have produced equivocal results (Bullard et al., Se . Oncol.. 13:94-109 (1986); Frank and Tribolet, Neurosurg. Rev.. 9:31-37 (1986)).

In an animal study of gliomas, immune rat cytotoxic T cells primed ex vivo against a rat glioma were administered intravenously to rats with intracerebral glioma and induced regression of these brain tumors (Holladay et al., J. Neurosurg.. 77:757-762 (1992); Holladay et al., Neurosurg.. 30:499-504 (1992); Holladay et al., Neurosurg.. 31:528-533 (1992), each of which is incorporated herein by reference). Thus, passive immunization of glioma-bearing rats provided a therapeutic advantage in this study. However, due to the partially immunologically privileged location of a CNS tumor, methods for using active immunotherapy to inhibit or prevent the growth of primary tumors of the CNS and metastatic lesions within the CNS have not yet been developed.

Thus, there exists a need for an effective means to stimulate the immune response of a patient having a primary CNS tumor or metastatic lesions located within the CNS, such that the patient's immune system can prevent or inhibit the growth of the tumor cells. The present invention satisfies this need and provides, in addition, related advantages. SUMMARY OF THE INVENTION

The invention provides a composition comprising haplotype-matched tumor cells which have been genetically modified to express a cytokine gene and to a composition comprising autologous fibroblasts which have been genetically modified to express a cytokine gene and unmodified tumor cells. The invention also provides methods for using the haplotype-matched cytokine-secreting cells to stimulate an immune response against a tumor located in the central nervous system of a cancer patient. The invention further relates to a method of inhibiting or preventing the growth of tumor cells in the central nervous system of a patient by immunizing the patient with the haplotype-matched cytokine-secreting cells.

A cytokine expressed by a haplotype-matched genetically modified cell is secreted at the immunization site in an effective amount, which is defined as a level of cytokine that is sufficient to induce or augment a systemic anti-tumor immune response. The haplotype-matched genetically modified cell can be a tumor cell, which contains the appropriate tumor antigen required to induce an immune response. In addition, the haplotype matched- genetically modified cell can be an autologous fibroblast, in which case tumor antigen is provided by including unmodified tumor cells in the composition used to immunize the patient. Immunization can be either at the site of the tumor in the CNS or at a site other than the CNS. An effective amount of cytokine secretion does not result in unacceptable patient toxicity because the level of secreted cytokine does not significantly affect systemic cytokine concentrations.

Since the amount of cytokine secreted by the haplotype-matched genetically modified cells is sufficient to induce anti-tumor immunity but is too low to produce unacceptable patient toxicity, the present approach provides the benefit of localized cytokine administration without producing undesirable side effects. Furthermore, the continuous localized expression of a cytokine at the site of immunization more effectively augments an immune response against the patient's tumor cells as compared to intermittent cytokine injections. The disclosed invention also provides the advantage of localized immunization with the haplotype-matched genetically modified cells and, therefore, avoids the necessity of cumbersome intravenous infusions required for immunotherapy with cells activated ex vivo such as expanded population of tumor infiltrating lymphocytes (see, for example, Rosenberg et al.. New Engl. J. Med.. 323:570-578 (1990).

BRIEF DESCRIPTION OF THE DRAWING

Figure 1 shows schematic diagrams of retroviral vectors DC/ADA/R/IL-2 and GlNaCvi2.23.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides a composition comprising haplotype-matched tumor cells which have been genetically modified to express a cytokine gene and provides a composition comprising autologous fibroblasts which have been genetically modified to express a cytokine gene and unmodified tumor cells. The invention also provides methods for using the haplotype-matched cytokine-secreting cells to stimulate an immune response against a tumor located in the central nervous system of a cancer patient.

The invention further provides a method of inhibiting or preventing the growth of tumor cells in the central nervous system of a patient by immunizing the patient with the haplotype-matched cytokine-secreting cells. The invention contemplates, in part, the stimulation of a patient's immune response against a primary CNS tumor or metastatic lesions located within the CNS by (a) obtaining tumor cells having a haplotype which is matched to the patient's haplotype; (b) introducing into the haplotype-matched tumor cells a gene encoding a cytokine such as IL-2 and, if desired, a suicide gene such as the herpes simplex virus thymidine kinase gene (HSV-TK), wherein the cytokine gene product is expressed and secreted in an effective amount by the haplotype-matched tumor cells and wherein expression of the suicide gene can be induced if desired; (c) if desired, irradiating the tumor cells so as to prevent the cells from proliferating in vivo ; and (d) immunizing the patient with the haplotype-matched cytokine- secreting tumor cells, such that expression and secretion of the cytokine gene product stimulates the patient's immune response but does not produce unacceptable patient toxicity.

The invention further contemplates the stimulation of a patient's immune response against a primary CNS tumor or metastatic lesions located within the CNS by (a) obtaining autologous fibroblasts, which inherently have a haplotype that is matched to the patient's haplotype; (b) introducing into the autologous fibroblasts a gene encoding a cytokine such as IL-2 and, if desired, a suicide gene such as the herpes simplex virus thymidine kinase gene (HSV-TK) , wherein the cytokine gene product is expressed and secreted in an effective amount by the cytokine-secreting fibroblasts and wherein expression of the suicide gene can be induced if desired; (c) obtaining tumor cells, which provide a source of tumor antigen, (d) irradiating said tumor cells so as to prevent the cells from proliferating in vivo ; and (e) immunizing the patient with the cytokine-secreting fibroblasts and the irradiated unmodified tumor cells, such that expression and secretion of the cytokine gene product stimulates the patient's immune response but does not produce unacceptable patient toxicity.

As used herein, "gene" means a nucleotide sequence encoding a desired gene product such as a cytokine or an active fragment of a protein or peptide having cytokine activity. A "gene product," therefore, is a protein or a peptide, wherein the protein or peptide may be an active fragment of the protein or peptide as it is normally expressed in a cell. As used herein, an "active fragment" means that the peptide or protein has cytokine activity. Such activity can be readily determined using assays well known in the art and described herein.

The invention provides haplotype-matched cells which have been genetically modified to express a cytokine gene. As used herein, "haplotype-matched" means that a genetically modified cell such as a tumor cell and the patient being treated share one or more major histocompatibility locus haplotypes. For example, if it is determined that a patient with a glioma expresses the major histocompatibility locus HLA-A2 haplotype, the patient will be immunized with HLA-A2 glioma cells that have been genetically modified to express and secrete a cytokine gene product. The haplotype of the patient can be readily determined using methods well known in the art.

Haplotype-matched tumor cells can be autologous or allogeneic. For example, the cytokine-secreting cells can be autologous fibroblasts or tumor cells obtained from the patient. The autologous cells, which are grown in tissue culture and genetically modified, inherently are haplotype-matched to the patient.

In addition, since various HLA-A haplotypes are known to be present in the human population, a panel of genetically modified tumor cells can be created. A panel of such allogeneic tumor cells can express, for example, the various different HLA-A haplotypes present in a population. In addition, various panels can represent tumors of different histologic origin such as glioma, neuroblastoma and other primary CNS tumor cells as well as other non-CNS tumors such as lung carcinoma, breast carcinoma, melanoma and other tumors that metastasize to the CNS. Thus, the invention provides haplotype-matched cytokine-secreting cells useful for immunizing cancer patients expressing various haplotypes and having various types of tumors in the CNS.

As used herein, the term "genetically modified" means that the haplotype-matched cells have been subjected to recombinant DNA techniques such that the cells can express and secrete a cytokine gene that has been introduced into the tumor cells. Methods for introducing a cytokine gene into a cell are well known in the art and described below.

For immunization, the tumor cells are of the same histologic origin as the patient's tumor. Tumor cells having a desired haplotype can be obtained from established allogeneic cells lines or can be autologous cells obtained from the patient to be treated. Where the tumor cell is obtained from a patient, the tumor cells will be grown in culture using methods well known by one skilled in the art of tissue culture. For example, methods for culturing primary human glioblastoma cells have been described by Bigner et al., J. Neuropathol. Exp. Neurol.. 40:201-229 (1981), which is incorporated herein by reference. If desired, the cells can then be genetically modified using methods described herein or well known in the art. Alternatively, the tumor cells can remain unmodified and can be injected with cytokine-secreting fibroblasts to stimulate an immune response in a patient. Numerous cytokine genes have been cloned and are available for use in this protocol. For example, the genes encoding various interieukins, gamma-interferon and granulocyte-macrophage colony stimulating factor are available from the American Type Culture Collection (see ATCC/NIH Repository Catalogue of Human and Mouse DNA Probes and Libraries, 6th ed. , 1992). In addition, genes encoding cytokines, including interleukin-6, granulocyte colony stimulating factor and human stem cell factor, are available commercially (Amgen, Thousand Oaks, CA; see, for example, Patchen et al., Exptl. Hematol. , 21:338-344 (1993) and Broudy et al., Blood, 82:436-444 (1993), each of which is incorporated herein by reference) . Similarly, gene encoding various isoforms of TGF-β, including TGF-βl, TGF- β2, TGF-β3, TGF-β4 and TGF-β5, also are available to those in the art.

In addition, selectable marker genes such as the neomycin resistance (Neo^R) gene are available commercially and the use of such selectable marker genes is described, for example, in Sambrook et al., 1989. Incorporation of a selectable marker gene allows for the selection of tumor cells that have successfully received and express a desired gene.

A suicide gene can be incorporated into a haplotype-matched genetically modified tumor cell to allow for selective inducible killing of the tumor cell after stimulation of the immune response. As used herein, a

"suicide gene" means a gene, the expression of which can result in the death of the cell expressing the suicide gene when the cell is exposed to certain drugs. An example of a suicide gene useful in the invention is the HSV-TK gene.

A tumor cell induced to express a transferred HSV-TK gene is selectively killed when exposed to a drug such as acyclovir or gancyclovir. A suicide gene also can be a gene encoding a non-secreted cytotoxic polypeptide. A suicide gene can be attached to an inducible promoter and, when destruction of a haplotype-matched cytokine-secreting tumor cell is desired, an agent that induces the promoter can be administered such that expression of the cytotoxic polypeptide kills the haplotype-matched cytokine-secreting tumor cell. However, destruction of a haplotype-matched cytokine-secreting tumor cell is not mandatory and may not be desired.

Numerous methods are available for introducing a nucleic acid sequence into a cell in vitro (see Kriegler et al., 1990, and Sa brook et al., 1989). For example, an appropriate nucleic acid sequence can be inserted into an expression vector such as a plasmid or a viral vector, which is introduced into a cell using methods well known in the art such as transfection, transduction, electroporation and lipofection. Examples of useful viral vectors include adenovirus and adeno-associated vectors (see, for example, Flotte, J. Bioenerg. Biome b.. 25:37-42 (1993) and Kirshenbau et al., J. Clin. Invest. 92:381-387 (1993), each of which is incorporated herein by reference) . Vectors are particularly useful when the vector contains a promoter sequence, which can provide constitutive or inducible expression of a cloned nucleic acid sequence. Such vectors are well known in the art (see, for example, Methods in Enzvmology, Vol. 185, D.V. Goeddel, ed. (Academic Press, Inc., 1990)) and available from commercial sources (eg., Promega, Madison, WI) .

An effective method for transferring a gene or other nucleic acid sequence into a cell is by using retroviral gene transduction. When retroviruses are used for gene transfer, replication competent retroviruses theoretically can develop by recombination between the retroviral vector and viral gene sequences in the packaging cell line utilized to produce the retroviral vector. However, packaging cell lines in which the production of replication competent virus by recombination has- been reduced or eliminated can be used. In any case, all retroviral vector supernatants used to infect patient cells can be screened for replication competent virus by standard assays such as PCR and reverse transcriptase assays (see, for example, Rosenberg et al.. New Engl. J. Med.. 323:570- 578 (1990), which is incorporated herein by reference).

Retroviral vectors useful for expressing a cytokine can be constructed using methods well known in the art. For example, a retroviral vector expressing an IL-2 gene product, DC/AD/R/IL-2, was described by Gansbacher et al., Cane. Res., 50:7820-7825 (1990); Gansbacher et al., Blood, 80:2817-2825 (1992); Gastl et al. Cane. Res.. 52:6229-6236 (1992), each of which is incorporated herein by reference (see Figure 1). In addition, a cytokine- expressing retroviral vector, designated GlNaCvi2.23, was obtained from Genetic Therapy, Inc. (Gaithersburg, MD; see Figure 1) .

Prior to immunization, the tumor cells can be irradiated so as to prevent the tumor cells from proliferating in vivo . Approximately 10⁶ to 10⁷ genetically modified cytokine-secreting cells are required for each immunization. The number of cells, however, can be adjusted so as to provide a sufficient number of cells to secrete an effective amount of the cytokine. As used herein, an "effective amount" of a cytokine is an amount that induces the patient's immune response without producing unacceptable toxicity in the patient. For example, in the first patient treated using the disclosed method, transient erythema at immunization sites and tumor necrosis were not observed until the IL-2 dose exceeded 100 unit/24 hours. Thus, therapy can be initiated with transduced tumor cells that secrete this dose of IL-2. Since transduced cells typically secrete approximately 20- 40 units of IL-2/10⁶ cells/24 hours, initial immunization requires injection of approximately 5 x 10⁶ genetically modified cytokine-secreting cells. The appropriate number of cytokine-secreting cells along with unmodified tumor cells, if required, can be injected subcutaneously, intramuscularly or in any manner acceptable for immunization.

A nucleic acid sequence of interest also may be introduced into a haplotype-matched cell using methods which do not require the initial introduction of the nucleic acid sequence into a vector. For example, a nucleic acid comprising a cytokine gene and a selectable marker can be introduced into a cell using a cationic liposome preparation (Morishita et al., J. Clin. Invest., 91:2580-2585 (1993), which is incorporated herein by reference) . In addition, a nucleic acid can be introduced into a haplotype-matched cell using, for example, adenovirus-polylysine DNA complexes (see, for example, Michael et al., J. Biol. Chem. , 268:6866-6869 (1993), which is incorporated herein by reference) . Other methods of introducing a nucleic acid sequence into a tumor such that a gene contained within the nucleic acid can be expressed are well known and described, for example, in Methods in Enzvmology. Vol. 185, 1990).

The following examples are intended to illustrate but not limit the scope of the invention.

EXAMPLE I

PREPARATION OF HAPLOTYPE-MATCHED GENETICALLY MODIFIED CELLS

This example illustrates the methods used to culture glioblastoma cells and genetically modify the cells such that the tumor cells express and secrete a cytokine gene product. Establishment of Primary Human Glioblastoma Cell Lines

Methods have been developed that permit the establishment of over 50% of primary glioblastoma tumors in continuous cultures suitable for retroviral gene transfer (Bigner et al., 1981). Briefly, the patient's tumor was obtained from a clinically indicated surgical resection, minced and placed in Richter's zinc option media. An aliquot of the cells was centrifuged, washed in Richter's zinc option media, then cryopreserved as a "back-up" culture j-n a solution containing 10% dimethylsulfoxide and 50% fetal calf serum. A portion of the established tumor cell line was expanded for transduction with the IL-2 retroviral vector and for application in immune response monitoring assays.

The glioblastoma cell culture was prepared by transferring the tumor tissue to a 60 mm tissue culture plate and resecting from the normal brain and necrotic tissue a sample of "pure" tumor using sterile forceps, scissors and scalpel. The selected tumor pieces were diced with sterile scissors into the smallest pieces possible. Four ml of 0.4% collagenase in serum-free medium containing gentamicin 50 μg/ml was added to the tumor tissue in the tissue culture plate, which was then incubated for 1-4 hours at 37 °C in a CO- incubator. (For larger tumor samples, the tumor pieces are placed in a 100 mm tissue culture plate and 8 ml of medium is added, as described above) . The plates were checked hourly and the sample was worked up and down in a pipette to encourage dissociation and to assess the optimal time for further processing.

When the tumor cells were freely dissociated, the entire sample was transferred to a 50 ml tube for centrifugation. The plates were rinsed with serum-free medium to collect all cells. Centrifugation was performed in an IEC PR6 centrifuge at 1000 rpm for five minutes. The supernatant was aspirated and the pellet resuspended in Richter's zinc option culture medium with gentamicin 50 μg/ml in an amount appropriate to distribute the cells into the number of dishes adequate to accept 7 x 10⁶ cells/100 mm dish with 10 ml of medium containing 20% fetal calf serum as described (Bigner et al., 1981). The cells then were incubated at 37 °C in a 5% C0₂ incubator. Unless the culture medium became extremely acidic, the original medium was not changed before 48 hours to allow the cells to attach. Gentamicin-free medium was used for subsequent medium changes. As the tumor cells reached confluency, they were detached from the plates using trypsin and passaged at low split ratios of 1:1 or 1:2 until the cultures grew adequately. The HLA-A2 glioblastoma cell line obtained was designated GT9.

Cytogenetic and other cell line characterization studies are performed to identify, for example, p53, PDGF, EGFR and TGF-β genotypes and phenotypes. These studies are performed within the first 72-96 hours to determine the presence of malignant cells and are repeated at intervals of 20-30 passage levels and at the 70th passage level as the tumors are established. To avoid chromosome breaks from ordinary fluorescent light bulbs, Westinghouse F40G0 (Gold) bulbs are used in the laminar flow hoods and cell culture rooms in which the cultures are being established.

Panels of genetically modified tumor cell vaccines can be prepared using HLA-typed primary glioma cell cultures as described by Bigner et al. (1981). The cell panels can represent several different histologic types of tumor cells and can express HLA-A2 or HLA-A1 loci, which are expressed by approximately 40% and 25% of the North American population respectively. The availability of this panel of tumor cells having various haplotypes affords the opportunity to develop genetically modified whole cell vaccines matched at these loci for a significant proportion of the North American population. Studies in melanoma patients have indicated that the HLA-A2 locus is a dominant haplotype for tumor antigen presentation which can mediate MHC-restricted tumor destruction by cytotoxic T cells (Crowley et al., Cane. Res. , 50:492 (1990); Crowley et al, J. Immunol.. 146:1692-1699); Pandolfini et al.. Cane. Res.. 51:3164-3170 (1991)). Thus, autologous HLA-A2 tumor cells such as GT9 that have been genetically modified to express IL-2, for example, can be used to stimulate the immune response of a significant fraction of glioma patients.

Preparation of Primary Cultures of Autologous Fibroblasts:

Primary cultures of autologous cultures can be obtained using methods well known in the art. Fibroblasts can be obtained from a skin punch biopsy.

Transduction of the Primary Human Glioblastoma Tumor Cells With an IL-2-containing Retroviral Vector:

Standard retroviral gene transfer methods were used to transduce the glioblastoma cultures with the IL-2 retroviral vectors. Cultured tumor cells (5 x 10⁴ cells/10 cm plate) were incubated with supernatant from the appropriate packaging cell line in the presence of polybrene (8 mg/ml) as described by Xu et al.. Virology. 171:311-341 (1989) and by Miller and Rosman, BioTechnigues, 7:980 (1989), each of which is incorporated herein by reference. After 24 hr, the cells were washed, then cultured in medium containing 100-150 μg/ml of the neomycin analogue, G418, to select for transduced cells. The cells then were cultured for 48 hr in DMEM supplemented with 10% fetal calf serum (FCS) . Transfected cells were selected 10-14 days after selection with G418 was begun. The G418 resistant cells were tested for IL-2 gene expression by measuring IL-2 in the culture supernatant using the ELISA assay described below. Aliquots of the G418 resistant cells were stored at -70 °C until required for immunizations.

Similar methods were used to tranduce autologous fibroblasts obtained from the patient.

Measurement of IL-2 Expression:

Transduced cell culture supernatants were analyzed for IL-2 secretion levels employing commercially available enzyme linked immunosorbent assay (ELISA) kits containing antibodies specific for human IL-2 (Genzyme or

T Cell Sciences). Briefly, 96-well plastic microtiter plates coated with the primary antibody were incubated with the test sample, washed, then incubated with the appropriate secondary antiserum conjugated to peroxidase or alkaline phosphatase. The enzymatic reaction was developed using a chromogen substrate and the optical density read on a micro-ELISA plate reader. These kits contain substitution control antibodies and standard IL-2 solutions of known concentration to permit quantitation of IL-2 levels.

EXAMPLE II IMMUNIZATION OF A PATIENT WITH AUTOLOGOUS

IL-2-SECRETING CELLS

This example illustrates the effectiveness of treating a human patient with autologous glioma cells which have been genetically modified to express and secrete IL-2 and with a combination of autologous fibroblasts which have been genetically modified to express and secrete IL-2 and autologous irradiated, unmodified tumor cells. Patient History:

A glioblastoma multiforme (GBM) patient was treated with IL-2 gene therapy. The patient is a 52 year old female with GBM of the right temporal lobe diagnosed in December 1992. She was initially treated with surgical resection, conventional radiotherapy and PCV chemotherapy (procarbazine, CCNU and vincristine) . Nine months later, a second resection was performed for tumor recurrence. Tumor pathology revealed a GBM at re-resection. The patient's tumor progressed after experimental treatment with accutane and with Iodine-131-labeled anti-tenacin monoclonal antibody. Subsequently, the patient was treated with experimental stereotactic radiation therapy designed to encompass the site of tumor involvement.

IL-2 Gene Therapy:

IL-2 gene therapy was initiated in January 1993, approximately one year after the first tumor resection. The patient received nine subcutaneous immunizations at 2 to 4 week intervals with either autologous, irradiated IL-2 transduced tumor cells (GT9 cells, as described in Example I) or a mixture of irradiated unmodified tumor cells and IL-2-transduced fibroblasts. The treatment protocol is shown in Table 1.

Two IL-2 retroviral vectors were employed in this study. The retroviral vector, DC/AD/R/IL-2, utilized an adenosine deaminase promoter to drive IL-2 expression (Figure 1; see, also, Gansbacher et al. 1990, 1992; Gastl et al., 1992). The retroviral vector GlNaCvi2.23 employed a cytomegalovirus promoter • (Figure 1; Genetic Therapy, Inc., Gaithersburg, MD) .

Table 1 lists the transduced cell types and IL-2 doses administered for each immunization. IL-2 secretion Table i

EL-2 GENE THERAPY OF GLIOBLASTOMA TREATMENT SUMMARY

by the transduced cells in vitro was measured by ELISA. Tumor cells transduced with DC/AD/R/IL-2 or GlNaCvi2.23 expressed similar amounts of IL-2 in vitro (10-43 units IL-2/106 cells/24 hrs). However, fibroblasts transduced with the GlNaCvi2.23 vector secreted approximately 5-10 fold higher levels of IL-2 compared to those transduced with the DC/AD/R/IL-2 vector (Table 1). The total administered IL-2 dose ranged from 3 to 440 units/24 hrs. The total tumor cell dose for each immunization was 10⁷ cells, the dose being adjusted using unmodified tumor cells.

Clinical Course:

No significant adverse reaction was observed at the immunization sites and no treatment related abnormalities were observed during monitoring of the patient's complete blood count, serum chemistry and urine specimens. Transient, mild erythema lasting less than 24 hr was observed at the injection site following immunization at IL-2 doses greater than 100 units/24 hrs. Tamoxifen (2 x 80 mg/day) was administered beginning approximately 3 months after the first immunization.

Magnetic resonance imaging (MRI) scans were performed at approximately 4 week intervals during the first five months of treatment. The scans revealed modest changes in overall tumor size with waxing and waning of peritumoral edema associated with alterations in decadron doses (not shown) . The MRI scan performed six months after the initiation of treatment (4 weeks after the final and highest dose of IL-2 was administered) revealed marked tumor necrosis with significant peritumoral edema (not shown) .

Clinically, the MRI findings were associated with an exacerbation of the patient's baseline left-sided weakness. However, this weakness gradually has improved following administration of increased doses of decadron, which then were gradually tapered. Stereotactic intraventriculostomy was performed to relieve increased pressure in the left third ventricle. Cytological evaluation of cerebrospinal fluid revealed the presence of inflammatory cells without detectable tumor cells.

In summary, IL-2 gene therapy resulted in no significant toxicity at the sites of immunization and was associated with the generation of a cellular anti-glioma immune response (see below). Marked tumor necrosis was observed following the final IL-2 immunization dose. Thus, the results establish the potential therapeutic value of the disclosed method for inhibiting or preventing the growth of tumor cells in the CNS by stimulating the patient's immune response by immunization with haplotype- matched cytokine-secreting tumor cells.

Immune Responsiveness:

Peripheral blood mononuclear cells and serum samples from the patient were analyzed to assess the cellular and humoral anti-glioma cell immune response against autologous tumor cells.

To determine cell mediated immunity, standard chromium release assays were used. Briefly, peripheral blood mononuclear (PBM) cells were isolated by Ficoll- Hypaque density centrifugation of heparinized blood and were stimulated in vitro by incubating the cells with irradiated autologous tumor cells at various ratios of PBM:tumor cells in 96-well round-bottomed plates in the presence of IL-2 for 7 days. The cells then were washed and restimulated for six additional days. Target tumor cells were labelled overnight with 100 μCi of chromium-51 at 37 °C. The labelled cells were extensively washed and mixed with various numbers of effector cells in 96-well V- bottom plates. After a 4 hr incubation at 37 °C, the plates were centrifuged at 400 x g for 5 min and the radioactivity determined in a 100 μl aliquot of the culture supernatant. The percent specific lysis was calculated using the formula:

< (^cP^mexp ~ ^cP^mbkgd ) / (^{c m}total ^{" c}P^mbkgd > > ^X 100.

The results of the chromium release assay revealed that at a ratio of 30:1 peripheral blood mononuclear cells:tumor cells, the level of tumor cell cytolytic activity increased 3-4 fold above the baseline level following the third and subsequent immunizations (not shown) . These findings are consistent with the generation of a cellular anti-glioma cell immune response.

The humoral immune response was measured using indirect immunofluorescence to identify antitumor antibodies present in the patient's serum. No humoral response against the autologous tumor cells was observed.

EXAMPLE III

TREATMENT OF PATIENTS HAVING A TUMOR IN THE CNS USING HAPLOTYPE-MATCHED CYTOKINE-SECRETING CELLS

This example illustrates the general application of the claimed invention to patients having primary CNS tumors or metastatic lesions in the CNS.

Patient Selection:

Patients will have a histologically confirmed diagnosis indicating the presence of a primary CNS tumor or metastatic lesions present within the CNS. Patients with tumors that must be resected for therapeutic purposes or disclosed herein. Autologous fibroblasts and tumor cells can be cultured using methods as described above or otherwise known to one in the art.

However, the above-described patients as well as patients in which tumor cell samples are unavailable can be immunized with allogeneic haplotype-matched genetically modified tumor cells, so long as such tumor cells are of the same histologic origin as the patient's tumor. For example, where a patient with a HLA-A2 haplotype has a tumor of the glioma series, immunization can utilize genetically modified GT9 cells, as described in Example I. Other appropriate allogeneic haplotype-matched genetically modified tumor cells can be obtained from a panel of such tumor cells that have been established as continuously cultured cells.

Pretreatment Evaluation:

Standard pretreatment evaluations are performed as follows:

1) History and physical examination including a description and quantitation of disease activity and tissue-typing of the patient.

2) Performance Status Assessment 0 = Normal, no symptoms 1 = Restricted, but ambulatory

2 = Up greater than 50% of waking hours, capable of self-care

3 = Greater than 50% of waking hours confined to bed or chair, limited self-care

4 = Bedridden 3) Pretreatment laboratory analysis, including complete blood count, including differential count, platelet count, PT, PTT, glucose, BUN, creatinine, electrolytes, SGOT, SGPT, LDH, alkaline phosphatase, bilirubin, uric acid, calcium and total protein albumin.

Other analyses are performed as deemed appropriate, including urinalysis, serum complement levels and immunophenotyping of peripheral blood B cell and T cell subsets. In addition, pretreatment evaluations can include chest X-ray and other diagnostic studies including computerized tomography (CT) , magnetic resonance imaging (MRI) or radionuclide scans to document and quantify the extent of disease activity. Follow-up evaluations of these assessments are performed at regular intervals during the course of therapy (approximately every 1 to 3 months) to monitor the patient's response to therapy and to identify potential signs of toxicity, thus permitting adjustments in the number and distribution of immunizations.

Restrictions on Concurrent Therapy:

For optimal effects of this treatment, patients should receive no concurrent therapy which is known to suppress the immune system.

Treatment Protocol:

Each patient will receive subcutaneous immunizations with autologous or allogeneic haplotype- matched cytokine-secreting tumor cells, which can be genetically modified to express and secrete, for example, IL-2, and with genetically modified cytokine-secreting autologous fibroblasts and unmodified irradiated tumor cells. Prior to immunization, tumor cells will be irradiated with approximately 7000 rads of radiation, so as to render the tumor cells incapable of proliferation in vivo . Treatment will proceed essentially as described in Example II.

In general, a tumor biopsy is taken approximately two months prior to the initiation of immunization. The tumor cells are adapted to tissue culture and, if desired, genetically modified to express a cytokine gene. Cytokine- secreting tumor cells can be isolated and used for immunization. However, if autologous tumor cells are unavailable or cannot be adapted to grow in tissue culture, allogeneic haplotype-matched cytokine-secreting tumor cells can be used for immunizing the patient.

The patient is immunized subcutaneously with haplotype-matched cytokine-secreting tumor cells or with cytokine-secreting autologous fibroblasts and irradiated unmodified tumor cells at 1-4 week intervals, with adjustments to the immunization schedule made as required. Where immunization involves, for example, the use of IL-2- secreting cells, the level of IL-2 secreted at the site of immunization will be escalated from 100 units/24 hr early in the immunization schedule to 400 units/24 hr later in the schedule. The number of injected IL-2-secreting cells will remain relatively constant at approximately 1 x 10⁶ to 1 x 10⁷ tumor cells/immunization site by adding an appropriate number of irradiated unmodified tumor cells to the IL-2-secreting tumor cells required to secrete the appropriate level of IL-2 as determined by one skilled in the art of tumor immunotherapy. Multiple immunization sites can be used if it is deemed desirable to increase the IL-2 dose to the patient. The patient will be physically examined on each of the three consecutive days following immunization and physical and laboratory evaluations will be made at weekly intervals.

Alternatively, a patient may be treated at the site of the tumor in the CNS. For example, during a surgical procedure to remove a CNS tumor, haplotype-matched cytokine-secreting cells can be placed in the region from which the tumor was removed surgically. In most cases, immunization with cytokine-secreting tumor cells at the time of surgery will utilize allogeneic haplotype-matched genetically modified tumor cells selected from a panel of genetically modified tumor cells. However, if autologous tumor cells had been available prior to surgery, such autologous tumor cells can be genetically modified and used to immunize the patient at the site of the tumor in the CNS or unmodified tumor cells can be administered in combination with cytokine-secreting autologous fibroblasts. In addition to using stereotactic surgical procedures to place genetically modified cytokine-secreting cells at the site of a tumor within the CNS, ultrasound- or computerized tomography-directed fine needle insertion can be employed to introduce cytokine-secreting cells into the site of the tumor.

Dose Adjustments:

Immunizations using cytokine-secreting cells are administered at intervals of 1-4 weeks. The patient is physically examined on each of the three consecutive days following immunization and physical and laboratory evaluations will be made at weekly intervals. In addition, the immunoresponsiveness of the patient is determined using the assays described above, including, for example, assays to determine changes in the activity of the cellular immune response in the patient.

So long as no toxicity is observed, subsequent immunizations are administered at intervals of 1-4 weeks. The results of the cellular and humoral immunoresponsiveness assays and tumor monitoring studies can be used to optimize the treatment protocol as determined by one skilled in the art of tumor immunotherapy. Although toxic side effects are not expected to result from these immunizations, potential side effects are treated as described above.

Treatment of Potential Toxicity:

Unacceptable toxic side effects at the site of immunization were not observed in the patient treated as described in Example II and, therefore, are not expected to result from these immunizations. However, potential side effects of these immunizations can be treated as required. For example, if massive tumor cell lysis results, any resulting uric acid nephropathy, adult respiratory distress syndrome, disseminated intravascular coagulation or hyperkalemia will be treated using standard methods well known in the art. Local toxicity at the sites of immunization will be treated with either topical steroids and, if necessary, surgical excision of the injection site. Generalized hypersensitivity reactions such as "the chills," fever or rash will be treated symptomatically with antipyretics and antihistamines. Patients should not be treated prophylactically. Edema, arthralgia, lymphadenopathy or renal dysfunction can be treated using corticosteroids and/or antihistamines. Anaphylaxis will be treated by standard means such as administration of epinephrine, fluids and steroids.

Other Assays:

Provided that sufficient material is available for evaluation the following assays also are performed. Standard i munofluorescence flow cytometry procedures are useful to evaluate changes in the percentage of T-cells, natural killer cells and B-cells associated with cytokine gene therapy. Monoclonal antibodies specific for T cells (CD2, CD3, CD4, CD8) , natural killer cells (CD16, CD57, CD58) and B cells (CD19, CD20) can be used for these studies.

Briefly, Ficoll-Hypaque purified mononuclear cells are incubated with the primary antibody for 1 hr at room temperature, washed, then incubated with fluorochrome conjugated secondary antibody. The cells are washed, fixed and the percentage of positive cells are determined using a Coulter Epics 4 flow cytometer. Incubation of the cells with isotype-matched control antibody instead of the primary antibody is useful as a negative substitution control.

Standard immunohistological methods employing monoclonal antibodies specific for the hematopoietic cell subsets described above can be used to characterize the immune effector cell infiltrates observed in delayed-type hypersensitivity skin test biopsy sites. Methods for immunohistological evaluations of fresh frozen cryostat tissue sections are well known in the art.

EXAMPLE IV

ANIMAL STUDIES

The rat glioma tumor model described by Holladay et al., 1990, 1992, demonstrates the usefulness of the disclosed method of stimulating an immune response in a subject against the subject's tumor. Gliomas are produced in the rats, as described, and various treatment modalities are employed.

Briefly, glioma-bearing rats are treated with haplotype-matched glioma cells, which are genetically modified to express a cytokine gene, or with unmodified tumor cells and genetically modified cytokine-secreting autologous fibroblasts. Immunization is at a site other than the CΝS or at a site within the CΝS. The stimulation of a cellular and humoral immune response is determined as described above. In addition, the effect of treatment on the tumor is monitored by sacrificing rats at various times after initiating treatment and examining the gross and histological anatomy of the tumor. The ability of immunized animals to reject a subsequent tumor challenge also is determined.

Although the invention has been described with reference to the disclosed examples, it should be understood that various modifications can be made without departing from the spirit of the invention. Accordingly, the invention is limited only by the following claims.

Claims

CLAIMSWe claim:

1. A method for inhibiting or preventing the growth of tumor cells in the central nervous system of a patient comprising the stimulation of the patient's immune response against the tumor cells by immunizing said patient with haplotype-matched cells genetically modified to express and secrete a cytokine gene product.

2. The method of claim 1 wherein said immunization is at a site other than within the central nervous system.

3. The method of claim 1 wherein said haplotype- matched cells are autologous cells.

4. The method of claim 1 wherein said haplotype- matched cells are allogeneic cells.

5. The method of claim 1 wherein said haplotype- matched cells are tumor cells.

6. The method of claim 1 wherein said haplotype- matched cells are fibroblasts.

7. The method of claim 6 further comprising immunizing with unmodified tumor cells.

8. The method of claim 1 wherein said matched haplotype is HLA-A2.

9. The method of claim 5 wherein said cells are glioblastoma multiforme cells.

10. The method of claim 5 wherein said tumor cells are GT9 cells comprising HLA-A2 glioblastoma multiforme cells genetically modified to express and secrete a cytokine gene product wherein said cytokine gene product is interleukin-2.

11. The method of claim 1 wherein said cytokine is selected from the group of interleukin-1, interleukin-2, interleukin-3, interleukin-4, interleukin-5, interleukin-6, interleukin 7, granulocyte-macrophage colony stimulating factor, granulocyte colony stimulating factor, human stem cell factor and gamma interferon.

12. The method of claim 1 wherein said cytokine is interleukin-2.

13. The method of claim 1 wherein said cytokine gene is present in an expression vector.

14. The method of claim 13 wherein said expression vector contains a suicide gene.

15. The method of claim 13 wherein said expression vector is selected from the group of a retroviral vector, an adenovirus vector, an adenovirus- associated viral vector.

16. The method of claim 14 wherein said retroviral vector has a promotor causing sustained expression and secretion of an effective amount of a cytokine gene product.

17. The method of claim 1 wherein said genetic modification comprises introducing a nucleic acid sequence comprising said cytokine gene into said cell using a method selected from the group of adenovirus-polylysine DNA complex cytokine gene transfer, direct cytokine gene transfer and cationic liposome-mediated cytokine gene transfer.

18. A method for stimulating a patient's immune response to tumor cells located within the central nervous system comprising the steps of:

a. obtaining tumor cells which have a haplotype that is matched to a haplotype expressed by the patient;

b. introducing into said haplotype-matched tumor cells a gene encoding a cytokine, wherein said cytokine is expressed and secreted by said haplotype-matched tumor cells, and, if desired, a suicide gene such as the herpes simplex virus thymidine kinase gene, the expression of which is inducible; and

c. immunizing the patient with said haplotype-matched cytokine-secreting tumor cells, wherein said cells express and secrete an effective amount of the cytokine gene product which is sufficient to stimulate the immune response but does not produce unacceptable patient toxicity.

19. A method for stimulating a patient's immune response to tumor cells located within the central nervous system comprising the steps of:

a. obtaining fibroblasts from the patient;

b. introducing into said fibroblasts a gene encoding a cytokine, wherein said cytokine is expressed and secreted by said fibroblasts, and, if desired, a suicide gene such as the herpes simplex virus thymidine kinase gene, the expression of which is inducible;

c. obtaining tumor cells from said patient; and

d. immunizing the patient with said cytokine-secreting fibroblasts and tumor cells, wherein said cells express and secrete an effective amount of the cytokine gene product which is sufficient to stimulate the immune response but does not produce unacceptable patient toxicity.

20. The method of claim 18 or claim 19 wherein said immunization is at a site other than within the central nervous system.

21. The method of claim 18 or claim 19 wherein said cytokine is selected from the group of interleukin-1, interleukin-2, interleukin-3, interleukin-4, interleukin-5, interleukin-6, interleukin 7, granulocyte-macrophage colony stimulating factor, granulocyte colony stimulating factor, human stem cell factor and gamma interferon.

22. The method of claim 18 or claim 19 wherein said cytokine is interleukin-2.

23. A composition for increasing a patient's immune response to tumor cells located in the central nervous system comprising haplotype-matched tumor cells genetically modified to express and secrete a cytokine gene product.

24. A composition for increasing a patient's immune response to tumor cells located in the central nervous system comprising autologous fibroblasts genetically modified to express and secrete a cytokine gene product and tumor cells.

25. The composition of claim 23 or claim 24 wherein said cytokine is selected from the group of interleukin-1, interleukin-2, interleukin-3, interleukin-4, interleukin-5, interleukin-6, interleukin 7, granulocyte- macrophage colony stimulating factor, granulocyte colony stimulating factor, human stem cell factor and gamma interferon.

26. The composition of claim 23 or 24 wherein said cytokine is interleukin-2.

27. The composition of claim 23 or claim 24 wherein said cytokine gene product is expressed and secreted in an effective amount which is sufficient to stimulate the immune response but does not produce unacceptable patient toxicity.