Cancer immunotherapy using autologous tumor cells combined with cells expressing a membrane cytokine

This invention comprises cellular vaccines and methods of using them in cancer immunotherapy, particularly in humans. The vaccines comprise a source of tumor-associated antigen, and a cytokine-secreting cell line. Tumor antigen may be provided in the form of primary tumor cells, tumor cell lines or tumor extracts prepared from the subject. In certain embodiments of the invention, the cytokine-secreting line is a separate tumor line that is allogeneic to the patient and genetically altered so as to produce a cytokine at an elevated level. Exemplary cytokines are IL-4, GM-CSF, IL-2, TNF-.alpha., and M-CSF in the secreted or membrane-bound form. In these embodiments, the cytokine-producing cells provide immunostimulation in trans to generate a specific immune response against the tumor antigen. Vaccines may be tailored for each type of cancer or for each subject by mixing tumor antigen with a favorable number of cytokine-producing cells, or with a cocktail of such cells producing a plurality of cytokines at a favorable ratio.

FIELD OF THE INVENTION
 The present invention relates generally to the fields of cellular
 immunology and cancer therapy. More specifically, it relates to the
 generation of an anti-tumor immune response in a human by administering a
 cellular vaccine, comprising cells genetically altered to secrete a
 cytokine, in combination with a source of tumor antigen.
 BACKGROUND
 In spite of numerous advances in medical research, cancer remains a leading
 cause of death throughout the developed world. Non-specific approaches to
 cancer management, such as surgery, radiotherapy and generalized
 chemotherapy, have been successful in the management of a selective group
 of circulating and slow-growing solid cancers. However, many solid tumors
 are considerably resistant to such approaches, and the prognosis in such
 cases is correspondingly grave.
 One example is brain cancer. Each year, approximately 15,000 cases of high
 grade astrocytomas are diagnosed in the United States. The number is
 growing in both pediatric and adult populations. Standard treatments
 include cytoreductive surgery followed by radiation therapy or
 chemotherapy. There is no cure, and virtually all patients ultimately
 succumb to recurrent or progressive disease. The overall survival for
 grade IV astrocytomas (glioblastoma multiforme) is poor, with 50% of
 patients dying in the first year after diagnosis.
 A second example is ovarian carcinoma. This cancer is the fourth most
 frequent cause of female cancer death in the United States. Because of its
 insidious onset and progression, 65 to 75 percent of patients present with
 tumor disseminated throughout the peritoneal cavity. Although many of
 these patients initially respond to the standard combination of surgery
 and cytotoxic chemotherapy, nearly 90 percent develop recurrence and
 inevitably succumb to their disease.
 Because these tumors are aggressive and highly resistant to standard
 treatments, new therapies are needed.
 An emerging area of cancer treatment is immunotherapy. The general
 principle is to confer upon the subject being treated an ability to mount
 what is in effect a rejection response, specifically against the malignant
 cells. There are a number of immunological strategies under development,
 including: 1. Adoptive immunotherapy using stimulated autologous cells of
 various kinds; 2. Systemic transfer of allogeneic lymphocytes; 3.
 Intra-tumor implantation of immunologically reactive cells; and 4.
 Vaccination at a distant site to generate a systemic tumor-specific immune
 response.
 The first of the strategies listed above, adoptive immunotherapy, is
 directed towards providing the patient with a level of enhanced immunity
 by stimulating cells ex vivo, and then readministering them to the
 patient. The cells are histocompatible with the subject, and are generally
 obtained from a previous autologous donation.
 One approach is to stimulate autologous lymphocytes ex vivo with
 tumor-associated antigen to make them tumor-specific. Zarling et al.
 (1978) Nature 274:269-71 generated cytotoxic lymphocytes in vitro against
 autologous human leukemia cells. Lee et al. (1996) abstract,
 Gastroenterology conducted an in vitro mixed lymphocyte culture with
 inactivated leukemic blast cells and autologous lymphocytes, and generated
 effector T lymphocytes cytotoxic for a tumor antigen on autologous blast
 cells. An MHC D-locus incompatibility was thought to be necessary to
 provide proper help in the lymphocyte culture. Lesharn et al. (1984)
 Cancer Immunol. Immunother. 17:117-23 developed cytotoxic responses in
 vitro against murine thymoma cells by allosensitization.
 Gately et al. (1982) J Natl. Cancer Inst. 69:1245-54 found that 5 out of 9
 human glioma cell lines did not elicit allogeneic cytolytic lymphocyte
 responses in ex vivo cultures. However, if inactivated, allogeneic
 lymphocytes were provided as stimulator cells in the cultures,
 tumor-specific cytolytic T lymphocytes and non-specific non-T effectors
 were generated to 4 of the nonstimulatory lines. In U.S. Pat. No.
 5,192,537, Osband suggests activating a tumor patient's mononuclear cells
 by culturing them ex vivo in the presence of tumor cell extract and a
 non-specific activator like phytohemagglutinin or IL-1, and then treating
 the culture to deplete suppresser cell activity.
 Despite these experimental observations, systemic administration of ex
 vivo-stimulated autologous tumor-specific lymphocytes has not become part
 of standard cancer therapy.
 Autologous lymphocytes and killer cells may also be stimulated
 non-specifically. In one example, Fc receptor expressing leukocytes that
 can mediate an antibody-dependent cell-mediated cytotoxicity reaction are
 generated by culturing with a combination of IL-2 and IFN-.gamma. U.S.
 Pat. No. 5,308,626. In another example, peripheral blood-derived
 lymphocytes cultured in IL-2 form lymphokine-activated killer (LAK) cells,
 which are cytolytic towards a wide range of neoplastic cells, but not
 normal cells. LAK are primarily derived from natural killer cells
 expressing the CD56 antigen, but not CD3. Such cells can be purified from
 peripheral blood leukocytes by IL-2-induced adherence to plastic (A-LAK
 cells; see U.S. Pat. No. 5,057,423). In combination with high dose IL-2,
 LAK cells have had some success in the treatment of metastatic human
 melanoma and renal cell carcinoma. Rosenberg (1987) New Engl. J Med
 316:889-897. This strategy is labor-intensive, costly, and not suited to
 all patients. Schwartz et al. (1989) Cancer Res. 49:1441-1446 showed that
 A-LAK cells are superior to LAK cells at reducing lung and liver
 metastases of breast cancer in experimental animal models, but this was
 not curative and there were no long-term survivors.
 For examples of trials conducted using LAK in the treatment of brain
 tumors, see Merchant et al. (1988) Cancer 62:665-671 & (1990) J.
 Neuro-Oncol. 8:173-198; Yoshida et al. (1988) Cancer Res. 48:5011-5016;
 Barbaetal. (1989)J. Neurosurg. 70:175-182; Hayes et al. (1988) Lymphokine
 Res. 7:337-345; and Naganuma et al (1989) Acta Neurochir. (Wien)
 99:157-160. Another study proposes therapy for recurrent high-grade glioma
 using autologous mitogen-activated and IL-2 stimulated (MAK) killer
 lymphocytes, in combination with IL-2. Jeffes et al. (1991) Lymphokine
 Res. 10:89-94. While none of these trials was associated with serious
 clinical complications, efficacy was only anecdotal or transient.
 Induction of tumor-specific immunity in patients receiving such treatments
 has not been shown.
 Another form of adoptive therapy using autologous cells has been proposed
 based on observations with tumor-infiltrating lymphocytes (1IL). TILs are
 obtained by collecting lymphocyte populations infiltrating into tumors,
 and culturing them ex vivo with IL-2. Finke et al. (1990) Cancer Res.
 50:2363-2370 have characterized cytolic activity of CD4+ and CD8+ TIL in
 human renal cell carcinoma. TILs have activity and tumor specificity
 superior to LAK cells, and have been experimentally administered, for
 example, to humans with advanced melanoma. Rosenberg et al. (1990) New
 Engl. J Med. 323:570-578. The effector population within TILs may be
 cytotoxic T lymphocytes (CTL) which are primed to be tumor-specific in the
 host and are devoid of lytic granules, and become transformed into
 cytolytic lymphoblasts when stimulated in culture. Berke et al.(1988) J.
 Immunol. 129:303 ff. Unfortunately, TILs can only be prepared in
 sufficient quantity to be clinically relevant in a limited number of tumor
 types. These strategies remain experimental, especially in human therapy.
 The second of the strategies for cancer immunotherapy listed earlier is
 adoptive transfer of allogeneic lymphocytes. The rationale of this
 experimental strategy is to create a general level of immune stimulation,
 and thereby overcome the anergy that prevents the host's immune system
 from rejecting the tumor. Strausser et al. (1931) J. Immunol. Vol.127,
 No.1 describe the lysis of human solid tumors by autologous cells
 sensitized in vitro to alloantigens. Zarling et al. (1978) Nature
 274:269-71 demonstrated human anti-lymphoma responses in via following
 sensitization with allogeneic leukocytes. Kondo et al. (1984) Med
 Hypotheses 15:241-77 observed objective responses of this strategy in
 20-30% of patients, and attributed the effect to depletion of suppressor T
 cells. The studies were performed on patients with disseminated or
 circulating disease. Even though these initial experiments were conducted
 over a decade ago, the strategy has not gained general acceptance,
 especially for the treatment of solid tumors.
 The third of the immunotherapy strategies listed earlier is intra-tumor
 implantation. This is a strategy directed at delivering effector cells
 directly to the site of action. Since the transplanted cells do not
 circulate, they need not be histocompatible with the host. Intratumor
 implantation of allogeneic cells may promote the ability of the
 transplanted cells to react with the tumor, and initiate a potent graft
 versus tumor response.
 Kruse et al. (1990) Proc. Natl. Acad. Sci. U.S.A. 87:9577-9581 demonstrated
 that direct intratumoral implantation of allogeneic cytotoxic T
 lymphocytes (CTL) into brain tumors growing in Fischer rats resulted in a
 significant survival advantage over other populations of lymphocytes,
 including syngeneic CTL, LAK cells, adherent-LAK cells or IL-2 alone. Redd
 et al. (1992) Cancer Immunol. Immunother. 34:349-354 developed cytotoxic T
 lymphocytes specific for an allogeneic brain tumor in rats. The
 lymphocytes were specific for a determinant expressed only by the tumor,
 and were predicted to be useful for therapeutic purposes in vivo. Kruse et
 al. (1994) J. Neurooncol. 19:161-168 prepared CTLs from four MHC
 incompatible rat strains, and used them to treat Fischer rats bearing
 established 9L brain tumors. CTL were administered on a biweekly schedule,
 a different MHC incompatible CTL preparation being administered each time.
 Animals without tumor showed minimal localized brain damage. Those with
 tumors either showed: a) mononuclear cell infiltration, massive tumor
 necrosis beginning 2-4 days after treatment, and total tumor destruction
 by 15 days; or b) cellular infiltration, early tumor destruction, and then
 tumor regrowth progressing to death of the animal. Tumor regressor animals
 were resistant to intracranial rechallenge with viable tumor cells. Kruse
 et al. (1994). Intratumor CTL implants may optionally be combined with
 chemotherapy using cyclophosphamide. Kruse et al. (1993) J. Neurooncol.
 15:97-112.
 Despite the promise of intratumor implantation techniques, several caveats
 remain. First, implantation is frequently performed by surgical
 techniques, which may be too invasive for routine maintenance. Second, the
 strategy is directed at generating a local response, and may not be
 effective against metastases. Finally, the techniques remain unproved for
 use in human therapy.
 The fourth of the immunotherapy strategies listed earlier is the generation
 of an active systemic tumor-specific immune response of host origin. The
 response is elicited from the subject's own immune system by administering
 a vaccine composition at a site distant from the tumor. The specific
 antibodies or immune cells elicited in the host as a result will hopefully
 migrate to the tumor, and then eradicate the cancer cells, wherever they
 are in the body.
 Various types of vaccines have been proposed, including isolated
 tumor-antigen vaccines and anti-idiotype vaccines. Mitchell et al. (1993)
 Ann. N Y Acad. Sci. 690:153-166 have treated cancer patients with
 mechanical lysates from a plurality of allogeneic melanoma cell lines,
 combined with the adjuvant DETOXIF. These approaches are all based on the
 premise that tumors of related tissue type all share a common
 tumor-associated antigen. For patients with tumors that did not acquire
 expression of the antigen during malignant transformation, or that
 subsequently differentiated so as not to express it, none of these
 vaccines will be successful.
 An alternative approach to an anti-tumor vaccine is to use tumor cells from
 the subject to be treated, or a derivative of such cells. For review see,
 Schirrmacher et al. (1995) J. Cancer Res. Clin. Oncol. 121:487-489. In
 U.S. Pat. No. 5,484,596, Hanna Jr. et al. claim a method for treating a
 resectable carcinoma to prevent recurrence or metastases, comprising
 surgically removing the tumor, dispersing the cells with collagenase,
 irradiating the cells, and vaccinating the patient with at least three
 consecutive doses of about 10.sup.7 cells. The cells may optionally be
 cryopreserved, and the immune system may be monitored by skin testing.
 This approach does not solve the well-established observations that many
 tumors are not naturally immunogenic. Many patients from which tumors have
 been resected are either tolerant or unable to respond to their own tumor
 antigen, even when comprised in a vaccine preparation.
 Several ways of preparing autologous or syngeneic tumor cells have emerged
 that potentially enhance immunogenicity. Tumor cells may be combined with
 extracts of bacillus Calmette-Guerin (BCG) or the A60 mycobacterial
 antigen complex. Berd et al. (1990) J. Clin. Oncol. 8:1858-67; Maes et al.
 (1996) J. Cancer Res. Clin Oncol. 122:296-300. Tumor cells may be lysed by
 or mixed with vaccinia virus. Hersey et al.; Ito et al. Tumor cells may be
 incubated with the Newcastle Disease Virus (NDV). U.S. Pat. No. 5,273,745.
 Autologous tumor cells may also be conjugated to haptens like
 dinitrophenyl. U.S. Pat. No. 5,290,551.
 In another approach to increase immunogenicity, Guo and coworkers (WO
 95/16775) suggest that tumor cells be fused with membrane components of a
 second cell that has a greater immunogenic potential. Suitable cells are
 an activated antigen-presenting cell such as a B cell. The fusion partner
 cell may be genetically altered to express an MHC protein, adhesion
 protein, or a cytokine. Rat hepatocarcinoma cells lost tumorigenicity when
 fused with syngeneic B cells, and were capable of eliciting a T-cell
 response. Rats injected with the hybrid cells generated CD4+ and CD8+ T
 cells against subsequent challenge, or eradicated preexisting tumors via a
 CD8+ T cell mediated mechanism.
 In yet another approach, autologous or syngeneic tumor cells are
 genetically altered to produce a costimulatory molecule. Examples of
 costimulatory molecules include cell surface receptors, such as the B7-1
 costimulatory molecule or allogeneic histocompatibility antigens.
 Salvadori et al.(1995) Hum Gene Ther. 6:1299-1306; Plaksin et al. (1994)
 Int. J. Cancer 59:796-801; EP 56967.
 Other examples are secreted activators, including cytokines. For review
 see, Pardoll et al. (1992) Curr. Opin. Immunol. 4:619-23; Saito et al.
 (1994) Cancer Res. 54:3516-3520; Vieweg et al.(l 994) Cancer Res.
 54:1760-1765; Gastl et al. (1992) Cancer Res. 52:6229-6236; and WO
 96/07433). Tumor cells have been genetically altered to produce
 TNF-.alpha., IL-1, IL-2, IL-3, IL-4, IL-6, IL-7, IL-10, IFN-.alpha.,
 IFN-.gamma. and GM-CSF. Asher et al. (1991) J. Imunol. 146:3227-3234;
 Blankenstein et al. (1991) J. Exp. Med. 173:1047-1052; Karp et al. (1993)
 J. Imunol. 150:896-908; Douvdevani et al. (1992) Int. J. Cancer
 51:822-830; Cavallo et al. (1992) J. Immunol. Vol. 149: 3627-3635 No. 11 &
 (1993) Cancer Res. 53:5067-5070; Fearon et al. (1990) Cell 60:397-403;
 Gansbacher et al. (1990) J. Exp. Med. 172:1217-1224; Connoret al. (1993)J.
 Exp. Med. Vol. 177:1127-1134; Topalianetal. (1988)J. Clin, Oncol.
 6:838-853; McBride et al. (1992) Cancer Res. 52:3931-3937; Golumbek et al.
 (1989) Science 254:713 ff & (1991) Science 254:713-716; Tepper et al.
 (1989) Cell 57:503-512; Santin et al. (1995b); Santin et al. (1995c) Int.
 J. Gynecol. Cancer 5:401-410; Gynecol. OncoL 58:230-239; Santin (1996) Am.
 J. Obst. Gynecol. 174:633-639; Allione et al. (1994) Cancer Res.
 54:6022-6026; EP 538952; Belldegrun et al. (1993) J. Natl. Cancer Inst.
 85:207-216; Dranoffet al. (1993) Proc. Natl. Acad Sci. USA Vol.
 90:3539-3543.
 Golumbek et al. (1989) reported that mouse renal carcinoma cells inserted
 with a gene for IL-4 was strongly immunogeneic for systemic T cell
 immunity, and protected mice against a subsequent lethal challenge with
 unmodified, parental tumor cells. Induction of an immune response does not
 depend on inherent immunogenicity; cytokines like IL-2 induce a response
 that is protective against otherwise non-immunogenic adenocarcinoma cells,
 including distant metastases. Cavallo et al. 1991 & 1992. Antitumor
 immunity is intensified by a cancer vaccine that produces both GM-CSF and
 IL-4. Wakimoto et al. (1996) Cancer Res. 56:1828-33. The cytokine or
 cytokine combination may recruit or stimulate cells of the immune system,
 and thereby overcome the normal barrier to immunity. Certain cytokines
 also affect the expression of major histocompatibility molecules and
 intercellular adhesion molecules by cancer cells (Santin et al. 1995a,
 Int. J: Cancer 65:688-694), potentially improving immunogenicity.
 The experiments with transduced histocompatible tumor cells have been done
 chiefly in genetically restricted animal models, which are not directly
 equivalent to a heterogeneous human patent population. Colombo et al.
 (1995) Cancer Immunol. Immunother. 41:265-270. Not all cancer types are
 responsive to the same cytokines. There are concerns about injecting human
 patients with replication-competent tumor cells, particularly after
 genetic alteration. In addition, there is usually not enough time to
 genetically alter and grow up sufficient cells of the the patient to be
 treated for use in a vaccine.
 Blumbach (WO 96/05866) has suggested vaccines of live tumor cells
 transduced with: a) a gene coding for an immunostimulatory protein; b) a
 cytokine; and c) a thymidine kinase gene. The composition is provided as
 live cells which can grow in vivo and stimulate a response, and then be
 selectively killed via the thymidine kinase. The possibility of escape
 mutants is likely to be a subject of regulatory concern for this approach
 in human therapy. Golumbek et al. (1992) J. Immunother. 12:224-230 have
 shown that proliferating tumor cells with suicide genes can also survive
 toxin treatment when they exit the cell cycle temporarily or are
 sequestered pharmacologically.
 As an alternative, Cohen (WO 95131107) suggested that neoplastic disease
 can be treated with a cellular immunogen comprising allogeneic cells
 genetically altered to express one or more cytokines, and also to express
 tumor-associated antigens encoded by autologous genomic tumor DNA. In this
 approach, an allogeneic cell (exemplified as a mouse LM cell) is
 genetically altered to express: a) a cytokine; and b) a tumor-associated
 antigen autologous to the subject to be treated. Accordingly, the vaccine
 need not comprise live tumor cells.
 However, application of the Cohen invention to human subjects would require
 prior knowledge for each patient of a particular tumor-associated antigens
 expressed by the particular tumor. Many human cancers of widespread
 clinical interest do not have reliable commonly-shared markers. Once a
 relevant marker is identified for a particular patient, a cell line must
 be engineered accordingly, and cultured to the required density prior to
 treatment. Thus, each patient would become their own research and
 development project Since the immune response would be focused only at the
 particular tumor-associated antigen used, it may be less effective than
 one directed against the spectrum of antigen expressed by a complete tumor
 cell. Furthermore, the vaccine comprises a live genetically altered cell
 line, raising the concerns outlined earlier. Cohen demonstrated only a
 modest improvement in survival in the animal studies, and failed to
 provide any evidence that his formulation would be effective in human
 cancer patients.
 A suitable strategy for a human anti-tumor cellular vaccine has to contend
 with the following problems: a) heterogeneity amongst tumors (even tumors
 of the same type) in the display of tumor-associated antigens; b)
 heterogeneity in the immune response between individuals with regards to
 both antigens and cytokines; c) ethical and regulatory concerns about
 compositions that may be used in humans; and d) lack of development time
 in most clinical settings, limiting the ability to engineer new cell lines
 or otherwise tailor the vaccine to each patient.
 SUMMARY OF THE INVENTION
 This invention provides compositions and methods for eliciting an
 anti-tumor immune response in a human patient in need thereof The
 compositions of the invention are cells or cell mixtures in a compatible
 excipient, and are referred to herein as a vaccine or an immunogenic
 composition. They may be administered to patients either to treat or
 palliate a clinically detectable tumor, or for prophylaxis, particularly
 after surgical debulking, chemotherapy or radiation therapy of a
 previously detectable tumor. The compositions are typically administered
 at a location distant from the original tumor, with the objective of
 stimulating a systemic reactivity against the tumor. The reactivity may in
 turn eradicate or slow the development of tumor cells, either at the
 primary site, within metastases (if there are any), or both.
 Minimally, the vaccines of this invention comprise two components. The
 first is a source of tumor antigen, preferably a plurality of antigens,
 which is associated with the cancer for which the patient is at risk. A
 convenient source of tumor-associated antigen is tumor cells previously
 obtained from the patient, such as during surgical resection. The second
 component is a cytokine producing cell capable of stimulating the
 patient's immune system to produce an anti-tumor response.
 In one series of preferred embodiments, the cytokine producing cell is a
 cell from an allogeneic donor, typically a tumor cell and preferably a
 tumor cell of the same type as the subject being treated, that has been
 genetically altered to express the cytokine at an elevated level. A
 preferred source of tumor antigen is the patient, and the vaccine is
 typically assembled by mixing tumor cells from the patient (or antigen
 therefrom) with the allogeneic cytokine-producing cells.
 In another series of preferred embodiments, the cytokine producing cell is
 a cell that is autologous or syngeneic to the patient that has been
 genetically altered to produce a cytokine. Typically, the cell will be a
 cell obtained from the patients tumor (or its progeny) that was
 subsequently altered so as to produce an effective amount of the cytokine.
 In this form, the same cell provides both components necessary to evoke
 the desired response: i.e., both the tumor antigen and the stimulatory
 cytokine.
 Cytokines useful for expression by the cytokine-producing cells according
 to either of these series of embodiments include those that promote
 immunostimulation against the tumor antigen by any mechanism. Preferred
 and non-limiting examples are IL-4, GM-CSF, IL-2, TNF-.alpha., and M-CSF.
 In certain embodiments, the cytokine is primarily secreted by the cell. In
 other embodiments, the cytokine is produced by the cell as a transmembrane
 protein, and provides immunostimulation by a mechanism that may involve
 intercellular contact Transmembrane cytokines include those such as
 mM-CSF, that naturally occur in a transmembrane form, and cytokines that
 naturally occur in a secreted form that are engineered to incorporate a
 region that allows them to be retained in the cell membrane.
 Also embodied in this invention are compositions and methods for treating a
 neoplastic disease such as cancer, comprising administering any one of the
 compositions or vaccines of this invention, or eliciting an anti-tumor
 immunological response according to any one of the methods of this
 invention. The treatment is effective in palliating the disease condition
 according to any clinically acceptable criteria for improvement, such as
 inhibition of tumor growth, increase in life expectancy of the patient, or
 improvement in quality of life or performance activity score.
 Further embodiments of the invention include kits and methods for
 assembling the immunological and pharmaceutical compositions of this
 invention for use according to the descriptions provided in this
 disclosure.

DETAILED DESCRIPTION
 A central feature of the cellular vaccines of this invention is the use of
 multiple components that act in concert once inside the host to produce
 the desired effect. In other words, the strategy is more than just an
 injection of cancer cells.
 The strategy is a significant departure from previous approaches to cancer
 immunotherapy in humans. Several important embodiments of this invention
 differ from other compositions comprising cancer cells, in that it
 contains separately: a) tumor associated antigen; and b) a cytokine
 expressing cell line that acts in trans to induce a beneficial response
 against the antigen.
 Tumor antigen is preferably provided from a cancer cell or the progeny
 thereof, preferably a cell autologous to the subject to be treated,
 typically obtained from the subject either by surgical resection, biopsy,
 blood sampling, or other suitable technique. The cytokine-producing cell
 is from a different donor which is genetically altered and characterized
 ahead of time for properties relating to its ability to stimulate an
 enhanced immune response when used in a vaccine of this invention. The
 genetically altered cell is allogeneic to the subject being treated, and
 is typically the same type of cancer as is borne by the subject.
 A separately filed patent application shows that mixed lymphocytes
 implanted directly into a tumor bed limits or even reverses tumor growth.
 These experiments and results are described in a PCT patent application
 published as WO 96/29394 (corresponding to PCT/US96/0362 1), which is
 hereby incorporated herein by reference in its entirety. The effect on
 tumor mass appeared in part to be due to an active immunological reaction
 of host origin, which appears to be a long-lasting one. It was
 hypothesized that increased expression transplantation antigens stimulated
 by allogeneic lymphocytes in the implant resulted in the massive
 recruitment of lymphoid cells near the tumor site, and that certain of the
 recruited cells played a role in reacting specifically against the tumor.
 A significant element of this hypothesis is that the cells stimulating the
 host immune response (the mixed lymphocytes) are different from the source
 of tumor antigen, but lead to reactivity against tumor antigen.
 Observations of this kind contributed partly to the inspiration for
 additional vaccine compositions. A second-generation vaccine would
 encompass a number of improvements over previously disclosed compositions.
 Desirable improvements include:
 a composition that could stimulate an active response against a plurality
 of tumor-associated antigens in any subject treated;
 the ability to prepare a vaccine without preculturing of the subject's
 cells, preferably at the instant that tumor cells are available from the
 subject;
 the use of cells of minimal proliferative capacity;
 a well-defined and reproducible immunostimulatory capacity;
 the ability to tailor the immunostimulatory capacity to the patient, as
 required; and
 a capability to administer the vaccine at a site distant from the primary
 tumor, preferably with minimal invasiveness.
 In order to meet these requirements, it was decided that the
 immunostimulatory cells and the source of tumor antigen should be
 different. Cells genetically altered to produce cytokines are strongly
 immunostimulatory. When cells are obtained from a donor other than the
 subject, they can be genetically altered in advance, cloned to stabilize
 the characteristics, selected for high levels of expression, and further
 selected for an ability to express cytokines even after inactivation. This
 eliminates the need to culture each autologous cell line, and has the
 benefit of careful standardization and quality control. The fact that the
 immunostimulatory cells are typically HLA-incompatible is probably
 irrelevant, since their main role is to initiate an immunological reaction
 in the host, which can then mature after the immunostimulatory cells are
 depleted. HLA-incompatibility can even be an advantage in the
 immunostimulatory potential of the cells.
 Stimulation is provided to generate an immune reaction against tumor
 antigen as a bystander. When used in an implant setting, a nexus of tumor
 antigen is supplied by residual primary tumor cells. For a systemic or
 distally administered composition, it is necessary to provide the nexus of
 tumor antigen by mixing it into the preparation. Preferably, a plurality
 of tumor antigen associated with the subject's own tumor is used. This is
 conveniently provided by using cells obtained from the subject's tumor,
 progeny thereof, or an extract of either the primary tumor cells or their
 progeny. These cells may also be inactivated, since they generally do not
 need to proliferate to provide tumor antigen. Using autologous tumor cells
 confers the additional advantage of being HLA-compatible, meaning the
 cells may persist near the injection site or at another site of ongoing
 immunological activity, to assist in the maturation of the response.
 A hallmark of the cellular vaccines of the present invention is that the
 effect is substantially greater than is obtained using tumor cells alone,
 or tumor cells mixed with previously used adjuvants or cofactors.
 Interaction between the tumor cells and the stimulated lymphocytes of the
 vaccines is probably complex. While not wishing to be bound by theory, it
 is envisaged that the cytokine expressed by the genetically altered cell
 is effective in recruitment, activation, or stimulating the interaction of
 host immune cells. The recruited and stimulated host cells may then
 respond to atypical (but otherwise less immunogenic) components in the
 vicinity, including any antigens present upon or within or secreted by the
 autologous tumor cells. The cytokine-producing cells may also play a role
 in promoting antigen processing and presentation, or provide
 co-stimulation for antigen being presented. In addition, the
 cytokine-producing cells may also provide specific immunostimulation in
 cis for the tumor antigens expressed by the cytokine-producing cells.
 Accordingly, in certain preferred embodiments of this application, the
 cells used to generate the cytokine-producing cells are derived from a
 tumor type that is closely related to that of the subject being treated.
 An immunological response resulting from administration of the vaccine may
 comprise both humoral and cellular components, but a cellular response is
 especially preferred. Cellular immunity (either cytotoxic lymphocytes, or
 helper-inducer cells recruiting other effector mechanisms) are believed to
 be important in providing a specific effect against the cells of the
 target neoplasia The presence of an immunological response may be
 monitored by standard immunological techniques. However, in human therapy,
 a primary objective is an improvement in the clinical condition of the
 patient. Clinical outcome is therefore a superior assay for the
 effectiveness of the compositions and methods of this invention when
 directed towards cancer treatment.
 The present invention is superior to strategies used or suggested
 previously. Advantages of the vaccine compositions of this invention
 include the following:
 The vaccines improve the clinical condition or prognosis of human cancer
 patients, even though tumor cells residing in cancer patients are
 apparently poorly immunogenic on their own.
 Although the response is presumably mediated by a tumor-associated antigen,
 there is no need to confirm the presence of any particular antigen on the
 tumor of a treated subject. Use of patent's own tumor cells or an extract
 of such cells ensures a spectrum of relevant antigens.
 There is no need to genetically alter patients' cells, or use patients' DNA
 to genetically alter cells of the vaccine.
 The strategy is aimed at generating a long-lived systemic immune response,
 and may therefore be effective not only against the primary tumor, but
 also against metastatic cells sharing tumor antigen with the primary
 tumor.
 With the exception of the initial sampling of the tumor cells, the protocol
 may be performed with minimally invasive procedures. The vaccine
 compositions are preferably administered at a site distant from the tumor.
 Subcutaneous routes of administration are preferred.
 A particularly beneficial feature of certain vaccines of the invention is
 the fact that vaccine compositions can be tailored to particular cancer
 types, clinical features, and even to an individual subject, as necessary.
 This is important where different tumor types respond to different
 cytokines and cytokine mixtures. For example, one tumor type can respond
 more frequently to IL-4 in combination with GM-CSF, whereas another tumor
 can respond to IL-4 in combination with TNF-.alpha.. Accordingly, a
 vaccine for the first tumor type is prepared by mixing cells genetically
 altered to express IL-4, and cells genetically altered to express GM-CSF
 with tumor-associated antigen from the subject to be treated. A vaccine
 for the second type is prepared by mixing cells expressing IL-4 with cells
 expressing TNF-.alpha. and tumor-associated antigen. There is no need to
 genetically alter a cell line to express multiple cytokines (although this
 is included in the invention) since lines expressing different cytokines
 may be combined. In another example, one tumor type can respond slightly
 better to IL-4 and GM-CSF at a 2:1 molar ratio, while another can respond
 slightly better to IL-4 and GM-CSF at a 1:2 molar ratio. The cells can be
 mixed together in a suitable proportion to provide a molar ratio suited
 for the tumor being treated. In a third example, the ratio of
 cytokine-secreting cells to tumor antigen or autologous tumor cells is
 also adjusted according to the tumor being treated.
 Fine-tuning of the components of the vaccine can be done according to
 previous observations on the effectiveness of the vaccine in various
 clinical settings, in the context of features of the tumor in the subject
 being treated. A principal feature is the type of cancer being treated,
 with optional secondary features including, but not limited to, the
 location of the tumor in the body, staging, invasiveness, morphological
 features, results of biochemical tests for antigen expression or genetic
 alteration conducted on patient's serum or a tumor sample, clinical
 features, and response to previous therapy.
 Fine-tuning the vaccine is an added benefit of the nature of the
 composition, but is not required. Many combinations of cytokine-producing
 cells and autologous tumor cells are effective, and are encompassed by the
 claimed invention. Effective combinations are readily determined by a
 practitioner of ordinary skill in the art by following the guidelines
 provided herein. The availability of a plurality of allogeneic
 cytokine-producing cells for admixing into a vaccine considerably
 facilitates not only the adjustment of the composition in accordance with
 previous experience, but also the initial testing of various potential
 combinations.
 Other embodiments of this invention involve genetically altering a
 patient's own tumor cells so as to produce a stimulatory cytokine,
 especially a membrane-bound cytokine. The same cell thus provides both the
 cytokine and the tumor antigen. These cells can be administered alone, or
 are optionally mixed with allogeneic cells producing additional cytokines
 in order to increase stimulation, again providing an opportunity to
 fine-tune the relative amount and mixture of cytokines provided.
 A further description of preferred ways to prepare and use the vaccine
 compositions of this invention are provided in the sections that follow.
 Definitions
 The terms "vaccine", "immunogen", or "immunogenic composition" are used
 herein to refer to a compound or composition, as appropriate, that is
 capable of conferring a degree of specific immunity when administered to a
 human or animal subject. As used in this disclosure, a "cellular vaccine"
 or "cellular immunogen" refers to a composition comprising at least one
 cell population, which is optionally inactivated, as an active ingredient.
 The vaccines, immunogens, and immunogenic compositions of this invention
 are active vaccines, which means that they are capable of stimulating a
 specific immunological response (such as an anti-tumor antigen or
 anti-cancer cell response) mediated at least in part by the immune system
 of the host individual. The immunological response may comprise antibody,
 immunoreactive cells (such as helper/inducer or cytotoxic cells), or any
 combination thereof, and is preferably directed towards an antigen that is
 present on a tumor towards which the treatment is directed. The response
 may be elicited or restimulated in a subject by administration of either
 single or multiple doses. Nothing further is required of a composition in
 order for it to qualify as a vaccine, unless otherwise specified.
 A compound or composition is "immunogenic" if it is capable of either: a)
 generating an immune response against an antigen (such as a tumor antigen)
 in a naive individual; or b) reconstituting, boosting, or maintaining an
 immune response in an individual beyond what would occur if the compound
 or composition was not administered. A composition is immunogenic if it is
 capable of attaining either of these criteria when administered in single
 or multiple doses.
 "Stimulating" an immune or immunological response refers to administration
 of a compound or composition that initiates, boosts, or maintains the
 capacity for the host's immune system to react to a target substance, such
 as a foreign molecule, an allogeneic cell, or a tumor cell, at a level
 higher than would otherwise occur. Stimulating a "primary" immune response
 refers herein to eliciting specific immune reactivity in a subject in
 which previous reactivity was not detected; for example, due to lack of
 exposure to the target antigen, refractoriness to the target, or immune
 suppression. Stimulating a "secondary" response refers to the
 reinitiation, boosting, or maintenance of reactivity in a subject in whom
 previous reactivity was detected; for example, due to natural immunity,
 spontaneous immunization, or treatment using one or several compositions
 or procedures.
 A "cell line" or "cell culture" denotes higher eukaryotic cells grown or
 maintained in vitro. "Progeny" of a cell include any cells formed by cell
 division of a progenitor, either in vivo or in vitro. It is understood
 that the descendants of a cell may not be completely identical (either
 morphologically, genotypically, or phenotypically) to the parent cell.
 "Inactivation" of a cell is used herein to indicate that the cell has been
 rendered incapable of cell division to form progeny. The cell may
 nonetheless be capable of response to stimulus, or biosynthesis and/or
 secretion of cell products such as cytokines. Methods of inactivation are
 known in the art. Preferred methods of inactivation are treatment with
 toxins such as mitomycin C, or irradiation. Cells that have been fixed or
 permeabilized and are incapable of division are also examples of
 inactivated cells.
 "Genetic alteration" refers to a process wherein a genetic element is
 introduced into a cell other than by mitosis or meiosis. The element may
 be heterologous to the cell, or it may be an additional copy or improved
 version of an element already present in the cell. Genetic alteration may
 be effected, for example, by transducing a cell with a recombinant plasmid
 or other polynucleotide through any process known in the art, such as
 electroporation, calcium phosphate precipitation, or contacting with a
 polynucleotide-liposome complex. Genetic alteration may also be effected,
 for example, by transduction or infection with a DNA or RNA virus or viral
 vector. It is preferable that the genetic alteration is inheritable by
 progeny of the cell, but this is not necessarily required for the practice
 of this invention, particularly when altered cells are used in a
 pharmaceutical composition without further proliferation. A cell is
 described as "genetically altered" if it has itself been subjected to
 genetic alteration, or if it is the progeny of a cell that was subjected
 to genetic alteration, providing it retains the alteration of the
 progenitor. A cell is said to be "inheritably altered" if a genetic
 alteration is present which is inheritable by progeny of the altered cell.
 The terms "tumor cell" or "cancer cell", used either in the singular or
 plural form, refer to cells that have undergone a malignant transformation
 that makes them pathological to the host organism. Primary cancer cells
 (that is, cells obtained from near the site of malignant transformation)
 can be readily distinguished from non-cancerous cells by well-established
 techniques, particularly histological examination. The definition of a
 cancer cell, as used herein, includes not only a primary cancer cell, but
 any cell derived from a cancer cell ancestor. This includes metastasized
 cancer cells, and in vitro cultures and cell lines derived from cancer
 cells.
 The term "tumor-associated antigen" or "TAA" is used herein to refer to a
 molecule or complex which is expressed at a higher frequency or density by
 tumor cells than by non-tumor cells of the same tissue type.
 Tumor-associated antigens may be antigens not normally expressed by the
 host; they may be mutated, truncated, misfolded, or otherwise abnormal
 manifestations of molecules normally expressed by the host; they may be
 identical to molecules normally expressed but expressed at abnormally high
 levels; or they may be expressed in a context or milieu that is abnormal.
 Tumor-associated antigens may be, for example, proteins or protein
 fragments, complex carbohydrates, gangliosides, haptens, nucleic acids, or
 any combination of these or other biological molecules. Knowledge of the
 existence or characteristics of a particular tumor-associated antigen is
 not necessary for the practice of the invention.
 A protein such as a cytokine is referred to as a "transmembrane" protein if
 it normally remains stably associated in the membrane of the cell in which
 it is produced. The term does not require any particular configuration of
 the protein in the lipid bilayer of the membrane.
 As used herein, "treatment" refers to clinical intervention in an attempt
 to alter the natural course of the individual or cell being treated, and
 may be performed either for prophylaxis or during the course of clinical
 pathology. Desirable effects include preventing occurrence or recurrence
 of disease, alleviation of symptoms, diminishment of any direct or
 indirect pathological consequences of the disease, preventing metastasis,
 lowering the rate of disease progression, amelioration or palliation of
 the disease state, and remission or improved prognosis.
 The "pathology" associated with a disease condition is anything that
 compromises the well-being, normal physiology, or quality of life of the
 affected individual. This may involve (but is not limited to) destructive
 invasion of affected tissues into previously unaffected areas, growth at
 the expense of normal tissue function, irregular or suppressed biological
 activity, aggravation or suppression of an inflammatory or immunological
 response, increased susceptibility to other pathogenic organisms or
 agents, and undesirable clinical symptoms such as pain, fever, nausea,
 fatigue, mood alterations, and such other features as may be determined by
 an attending physician.
 An "effective amount" is an amount sufficient to effect a beneficial or
 desired clinical result, particularly the generation of an immune
 response, or noticeable improvement in clinical condition. An immunogenic
 amount is an amount sufficient in the subject group being treated (either
 diseased or not) to elicit an immunological response, which may comprise
 either a humoral response, a cellular response, or both. In terms of
 clinical response for subjects bearing a neoplastic disease, an effective
 amount is amount sufficient to palliate, ameliorate, stabilize, reverse or
 slow progression of the disease, or otherwise reduce pathological
 consequences of the disease. An effective amount may be given in single or
 divided doses. Preferred quantities and cell ratios for use in an
 effective amount are given elsewhere in this disclosure.
 An "individual" or "subject" is a vertebrate, preferably a mammal, more
 preferably a human. Non-human mammals include, but are not limited to,
 farm animals, sport animals, and pets.
 General Techniques
 The practice of the present invention will employ, unless otherwise
 indicated, conventional techniques of molecular biology, microbiology,
 cell biology, biochemistry and immunology, which are within the skill of
 the art. Such techniques are explained fully in the literature, such as,
 "Molecular Cloning: A Laboratory Manual", second edition (Sambrook et al.,
 1989); "Oligonucleotide Synthesis" (M. J. Gait, ed., 1984); "Animal Cell
 Culture" (R. I. Freshney, ed., 1987); "Methods in Enzymology" (Academic
 Press, Inc.); "Handbook of Experimental Immunology" (D. M. Weir & C. C.
 Blackwell, eds.); "Gene Transfer Vectors for Mammalian Cells" (J. M.
 Miller & M. P. Calos, eds., 1987); "Current Protocols in Molecular
 Biology" (F. M. Ausubel et al., eds., 1987); "PCR: The Polymerase Chain
 Reaction", (Mullis et al., eds., 1994); "Current Protocols in Immunology"
 (J. E. Coligan et al., eds., 1991). See also Gately et al., Lee et al.,
 and Zarling et al. (infra) for examples of techniques in mixed lymphocyte
 cultures.
 General procedures for the preparation and administration of pharmaceutical
 compositions are outlined in Remington's Pharmaceutical Sciences 18th
 Edition (1990), E. W. Martin ed., Mack Publishing Co., PA.
 All patents, patent applications, articles and publications mentioned
 herein, both supra and infra, are hereby incorporated herein by reference.
 Preparation of Cellular Vaccines
 The cellular vaccines of this invention are typically assembled by
 preparing each cell population or equivalent thereof in an appropriate
 fashion, combining the components, and optionally coculturing or storing
 cell mixtures before administration to a subject.
 Tumor-associated Antigen:
 The source of tumor-associated antigen is most usually a tumor cell or cell
 line that is close in phenotype to that for which the patient is being
 treated. Tumors from the same tissue type and with similar histological
 characteristics tend to share tumor-associated antigens. While the
 complete spectrum of antigens may vary between individual tumors, there is
 a substantial probability that at least one will be shared. Preferably,
 the tumor cells are histocompatible with the subject to be treated.
 Generally, when it is possible to obtain tumor cells in advance from the
 subject to be treated, these cells are preferred as more likely to bear a
 full complement of relevant tumor-associated antigens. Circulating tumors
 such as leukemias and lymphomas may be readily sampled from peripheral
 blood. Otherwise, tumor cells are generally sampled by a surgical
 procedure, including but not limited to biopsy, or surgical resection or
 debulking. Tumor cells may also be collected from metastatic sites. Solid
 tumors may be dissociated into separate cells by physical manipulation
 optionally combined with enzymatic treatment with such proteases as
 collagenase and the like. The cells are then transferred into fresh
 medium. Cells may be stored until further use, for example, by freezing in
 liquid N.sub.2. Optionally, and especially when the original tumor mass is
 small, it is preferable to expand the tumor cell population to ensure an
 adequate supply. Cells are cultured in a growth medium suitable for
 propagation, optionally supplemented with growth factors.
 Preferably, a stable cell population comprising features of the tumor cells
 is obtained without further transformation, although transformation is
 permissible where required. The cell population can be optionally cloned
 to enhance its stability or refine its characteristics, although this is
 generally not necessary. Conditions for reliably establishing short-term
 cultures and obtaining at least 10.sup.8 cells from a variety of tumor
 types is described in Dillman et al. (1993) J. Immunother. 14:65-69. If
 possible, the original tumor cell preparation is used without
 proliferation, since it is possible that a critical tumor antigen will be
 lost through the proliferative process.
 Cancer cells or cell lines obtained as described may be combined directly
 with the other components of the vaccine. However, it is preferable to
 inactivate the cancer cells to prevent further proliferation once
 administered to the subject. Any physical, chemical, or biological means
 of inactivation may be used, including but not limited to irradiation
 (preferably with at least about 5,000 cGy, more preferably at least about
 10,000 cGy, even more preferably at least about 20,000 cGy); or treatment
 with mitomycin-C (preferably at least 10 .mu.g/mL; more preferably at
 least about 50 .mu.g/mL).
 Cancer cells for use as a tumor antigen source may alternatively be fixed
 with such agents as glutaraldehyde, paraformaldehyde, or formalin. They
 may also be in an ionic or non-ionic detergent, such as deoxycholate or
 octyl glucoside, or treated, for example, using Vaccinia Virus or
 Newcastle Disease Virus. If desired, solubilized cell suspensions may be
 clarified or subject to any of a number of standard biochemical separation
 procedures to enrich or isolate particular tumor-associated antigens or
 plurality of antigens. Preferably, tumor antigen associated with the outer
 membrane of tumor cells, or a plurality of tumor associated antigens is
 enriched. The degree of enrichment may be 10-fold or more preferably
 100-fold over that of a whole-cell lysate. Isolated antigens, recombinant
 antigens, or mixtures thereof may also be used. Before combination with
 other components of the vaccine, the tumor antigen preparation is depleted
 of the agent used to treat it; for example, by centrifuging and washing
 the fixed cells, or dialysis of the solubilized suspension. Preparation of
 tumor antigen, particularly beyond inactivation of the source tumor cell,
 may be viewed as optional and unnecessary for the practice of the
 embodiments of the invention, unless specifically required.
 Cytokine-producing Cells:
 The cellular vaccines of this invention also comprise a second cell
 population, of which at least a portion are cells producing a soluble or
 membrane-bound factor capable of potentiating an immunological response
 against the tumor-associated antigen or autologous cell of the vaccine.
 Any cytokine or chemokine may be used for this purpose, especially those
 that have amongst their biological activities one or more of the
 following: a) the ability to recruit, enhance proliferation, enhance
 cytokine secretion by, or otherwise activate cells of the lymphocyte
 lineage; b) the ability to enhance uptake into antigen-presenting cells,
 the subsequent processing and display of antigen, or the concurrent
 production of cytokines; c) the ability to enhance display of
 histocompatibility antigens; d) the ability to enhance display of
 tumor-associated antigen by tumor cells; e) the ability to recruit other
 cells or soluble components that may participate in inflammation; or f)
 any other effect that results in a localized immune stimulation. The
 effect or effects can be measured in vitro according to standard
 immunological techniques, but should come into play in sufficient
 proximity to the tumor-associated antigen to provide an immunostimulatory
 effect that is at least partly specific for the antigen. A cytokine
 capable of mediating a plurality of the above-listed effects are
 particularly preferred.
 Preferred cytokines include, but are not limited to, tumor necrosis
 factors, exemplified in TNF-.alpha.; interleukins, exemplified in IL-2,
 IL-4, IL-6, IL-7, and IL-10; interferons, exemplified in IFN-.alpha. and
 IFN-.gamma.; hematopoetic factors; and colony stimulating factors,
 exemplified in GM-CSF and M-CSF. Different cytokines are more effective in
 certain cancers than others, and may vary between different cancers and
 patient groups. TNF and IL-2 are effective in cancers like adenocarcinoma
 in syngeneic animal vaccines, but less effective in ovarian or brain
 cancer, while M-CSF is especially potent in syngeneic animal vaccines for
 brain cancer.
 Amongst the possible cytokines that can be used with this invention, GM-CSF
 and M-CSF are especially preferred because of their important role in the
 maturation and function of specialized antigen-presenting cells. This is
 believed to be important because many tumor cells, such as those of
 epithelial origin, do not express detectable MHC class II molecules. IL-4
 is also preferred, as a pluripotent cytokine endowed of a broad range of
 stimulating activities on both B and T lymphocytes, as well as on
 hematopoietic cells. Its roles include the recruitment and activation of
 CD4+ antigen-presenting cells, as well as induction of cytotoxic T
 lymphocytes. TNF-.alpha. is a fourth cytokine which is preferred, in part
 because of its broad range of effects in the immune and inflammatory
 responses. Amongst cytokine combinations, the combination of GM-CSF and
 IL-4 is especially preferred. Embodiments of the invention with both of
 these cytokines are vaccines comprising autologous cancer cells and
 allogeneic cells genetically altered to express both GM-CSF and IL-4, or
 even more preferably vaccines comprising autologous cancer cells,
 allogeneic cells genetically altered to express GM-CSF, and different
 allogeneic cells genetically altered to express IL-4.
 The majority of cytokine produced by the cells used in this invention may
 be secreted from the cells, or present on the outer membrane of the cells.
 Where the cytokine has a local immunostimulatory effect, it can be
 preferable that it be primarily attached to the cell membrane to keep it
 in the vicinity of bystander tumor antigen comprised in the vaccine. Where
 the cytokine has a recruitment effect, it can be preferable that it be
 primarily secreted. As a third option, the cytokine can be synthesized by
 the cell in both membrane-associated and secreted form. As illustrated in
 Example 5, the preferable form of a particular cytokine can be determined
 by simple side-by-side comparison. M-CSF may be used in either form, and
 in certain vaccine compositions may be more effective in the
 membrane-associated form. While not wishing to be bound by theory, it is
 possible that the membrane-associated form creates a bridge between the
 allogeneic tumor cell and antigen-presenting cells or responder
 lymphocytes; in effect a forced antigen presentation. M-CSF may have an
 advantage over many other cell-surface receptor ligands in this regard,
 because of an ability to simultaneously bridge cells, and provide a
 stimulatory signal through its cytokine effect
 Other cytokines that have both of these properties may be particularly
 effective in tumor vaccine compositions, and cells of the vaccine are
 preferably altered to express them in the membrane-associated form. Where
 particular cytokines have potent immunostimulatory activity but do not
 occur naturally in a membrane-bound form, it is possible to create a
 membrane-bound form as a fusion protein. Allogeneic cells are genetically
 altered with a vector comprising a cytokine encoding region and a
 transmembrane region in the same open reading frame, the transmembrane
 region being either upstream or downstream from the cytokine encoding
 region and optionally separated by an in-frame spacer region. The
 transmembrane region may be modeled on other known transmembrane proteins,
 or be an artificially designed polypeptide segment with a high degree of
 lipophillicity.
 The protein and DNA encoding sequences of human IL-4 and TNF-.alpha. are
 known, and vectors comprising encoding sequences are available. For the
 IL-4 sequences and vectors, see U.S. Pat. No. 5,017,691 and EP 230107.
 Genetically altered CHO cells are described in U.S. Pat. No. 5,034,133.
 The use of IL-4 (either as the isolated recombinant or in a genetically
 altered cell) in treating solid tumors are described in U.S. Pat. No.
 5,382,427. TNT polypeptides, encoding sequences, vectors, and genetically
 altered host cells are described in U.S. Pat. No. 5,288,852, EP 155549,
 and U.S. Pat. No. 4,879,226. Variants of INF, which may also be used in
 this invention, are described in U.S. Pat. No. 4,677,063. Compositions
 comprising TNF-.alpha. and interferon are taught in EP 131789. Synergism
 of TNF and IL-4 in the inhibition of cancer cell growth is described in WO
 92/05805.
 Other cytokines and cytokine-encoding polynucleotides are described further
 in the example section below, or may be readily obtained through publicly
 available biological deposits, or may be prepared according to publicly
 available disclosures.
 The cell used to produce the cytokine for the vaccines of this invention is
 obtained from a different donor than the subject being treated. The donor
 is of the same species as the subject. Consequently, except in unusual
 circumstances, the cell is allogeneic to the subject. For the general
 practice of this invention, this definition is satisfied by at least one
 allelic difference at the amino acid level in a major histocompatibility
 complex (MHC) Class I or Class II antigen between the cytokine-secreting
 cell and the subject to be treated. Typically, a plurality of differences
 will be present in both Class I and Class II antigens will be present, and
 these differences will be recognizable either in an antibody-mediated
 tissue typing cytotoxicity test, or a mixed lymphocyte reaction between
 the cytokine-secreting cells and lymphocytes from the subject to be
 treated. Differences in Class I antigens are generally more relevant,
 since most cell types do not express Class II. In certain embodiments of
 this invention, the number of MHC differences is irrelevant, as long as
 the cells are allogeneic. In other embodiments of this invention, MHC
 differences, particularly Class I differences are preferred as a
 potentiating factor in immune stimulation. In this context, at least 2
 differences are preferred; at least about 3 differences are even more
 preferred. When using human cells, differences are especially preferred in
 the HLA-A -B and -C loci.
 The cell will also generally be a cell that can be maintained in culture
 for a large number of replications and genetically desired, if necessary.
 Typically, the cell will be a neoplastic cell, a malignantly transformed
 cell, or the progeny of such cells. Cells may be deliberately transformed
 into long-lived cell lines by any method, including, but not limited to,
 fusion with other cell lines, treatment with a chemical carcinogen, or
 infection with a suitable virus such as Epstein-Barr virus or oncogenic
 virus. More usually, the cell will be the progeny of a primary tumor
 occurring in the appropriate species, that has been established in ex vivo
 culture.
 In certain embodiments of this invention, tumor cells are used as the
 allogeneic cytokine-expressing cell, wherein the tumor is a different type
 from that of the subject being treated. This may provide additional
 bystander effect by providing a plurality of novel immunogenic antigens.
 In this context, the tumor cell is preferably selected so as to comprise a
 large proportion or particularly high level of an immunogenic epitope. In
 other embodiments of this invention, tumor cells are used from a tumor
 type similar or identical to that of the subject in terms of its tissue
 source, morphological characteristics, surface antigen expression,
 clinical manifestations, and any other relevant criteria. This is
 preferred when it is desirable to increase the probability that a
 tumor-associated antigen, or a plurality of such antigens may be
 overexpressed both on the cytokine-expressing tumor cell of the vaccine,
 and tumor cells in the subject being treated. Shared tumor-associated
 antigens may permit cis stimulation of therapeutically relevant immune
 reactivity.
 Cells may be stimulated to secrete cytokines at suitable levels according
 to any method known in the art, including, but not limited to, coculturing
 with other cells or treatment of the cells with the same or different
 cytokines.
 Most typically, in order to provide a high and reliable level of cytokine
 expression, the cells are genetically altered so as to synthesize the
 cytokine at an elevated level. It is recognized that certain cells such as
 lymphocytes and macrophages may already produce detectable levels of
 certain cytokines. "Elevated levels" of expression that occur as a result
 of genetic alteration exceed levels observed in cells not genetically
 altered or otherwise manipulated in the same way, but that are otherwise
 similar.
 Genetic alteration may be effected by any method known in the art.
 Typically, an encoding sequence for the desired cytokine is operatively
 linked to a heterologous promoter that will be constitutively or inducibly
 active in the target cell, along with other controlling elements and a
 poly-A sequence necessary for transcription and translation of the
 protein. The expression cassette thus composed is introduced into the cell
 by any method known in the art, such as calcium-phosphate precipitation,
 insertion using cationic liposomes, or using a viral vector tropic for the
 cells. Methods of genetic alteration are described in the patent
 publications cited in relation to some of the cytokines listed earlier.
 One preferred method is the use of adenovirus vectors. For example see,
 Graf et al. (1995) Soc. Neuroscience 21:838.5. Briefly, adenovial
 recombinant expression vectors prepared by genetic engineering of
 commercially available plasmids such as those supplied by Microbix,
 Canada. Suitable infection conditions and multiplicities of infection
 (MOI) may be determined in preliminary experiments using a reporter gene
 such as .beta.-galactosidase, and then used for cytokine transfer
 (Kammersheidt et al.). An advantage of using a viral vector is that the
 vector may first be replicated, and then an entire population of cells may
 be infected and altered. Accordingly, genetically altered cytokine
 secreting cells may be established as a cell line, or a freshly obtained
 cell isolate or cell culture is altered de novo just prior to use in a
 vaccine of this invention In the latter instance, preparation of the
 vaccine would additionally comprise the step of transducing a population
 of cells allogeneic to the intended recipient with a vector comprising an
 encoding region for a particular cytokine of interest. Transduction using
 adenoviral vectors and the like is especially preferred when it is
 desirable to achieve very high levels of cytokine expression by the
 genetically altered cells.
 An even more preferred preferred method of genetic alteration is the use of
 a retroviral vector comprising a suitable expression cassette.
 Non-limiting illustrations are provided in the example section below, and
 may also be found in Santin 1995b, 1995c & 1996. Although this approach
 may not achieve quite the same level of expression available in some other
 systems, a particular benefit is that the genetically altered cell is
 highly stable in the amount of cytokine produced. This means that the
 level of expression can be characterized exactly, and relied upon as a
 reagent composition through multiple passages and different storage
 conditions. In addition, genetically altered cells may be prepared which
 are capable of producing cytokine even after inactivation by irradiation.
 The levels of cytokine can be adjusted upwards, where necessary, simply by
 increasing the number of genetically altered cells in the dosage.
 As shown in Examples 1-4, tumor lines can be created using the LXSN
 retroviral vector that produce cytokine at a stable and reliable level
 through multiple cell divisions. Levels of cytokine secretion may be
 determined by immunoassay or bioassay. Cells with these properties are
 generally preferred, since they can be biochemically characterized and
 clinically tested in advance. Accordingly, it is generally preferable to
 clone genetically-altered cells and select high-producer clones.
 Supernatant of 1 x 106 cells/ml cultured in 10 ml medium for 48 hours at
 37.degree. C. may contain the following preferred levels of cytokines:
 IL-4, IL-2, TNF-.alpha., the secreted form of M-CSF, or most other
 cytokines of about the same molecular mass are preferably produced at
 least 500, more preferably at least about 1000, even more preferably at
 least about 2000 pg/mL. GM-CSF is preferably produced at least 100, more
 preferably at least about 200, even more preferably at least about 400
 pg/mL. Membrane-associated cytokines, where the majority produced by the
 cell, should be biosynthesized at a rate preferably 25%, more preferably
 50%, and even more preferably 100% of the range obtained for high-level
 producers of the secreted form.
 It is also highly desirable that a substantial proportion of
 cytokine-producing cells remain viable and be able to secrete the cytokine
 of interest after inactivation to prevent proliferation. Preferred
 treatments halt development of at least about 95%, or more preferably at
 least about 99% of the cells. Typically, when using irradiation, the
 levels required are 2,500 rads, more preferably 5,000 rads, even more
 preferably 10,000 rads, and still more preferably 20,000 rads. The cells
 preferably produce cytokine 2 days after irradiation at a rate that is at
 least about 10%, more preferably at least about 20%, more preferably at
 least about 50%, still more preferably at least about 100% of the
 pre-irradiated level, when standardized for viable cell number.
 The cytokine producing cells can also be modified in other ways, if
 desired. In particular, they can be genetically altered to express
 additional proteins, including but not limited to additional cytokines,
 additional tumor-associated antigens, or additional cell-surface markers,
 such as adhesion molecules like ICAM-1, histocompatibility antigens, or
 costimulation markers like the B-cell marker B7-1 or B7-2. Alternatively
 or in addition, they may be modified so as to produce multiple copies of
 the same or similar proteins, including multiple copies of the same
 cytokine in membrane-associated or secreted form, or both. Transduction
 for expression of multiple proteins or multiple protein copies may be
 conducted concurrently or sequentially. More than one genetic alteration
 may be viewed as optional, and is not required for the practice of this
 invention.
 Assembly of the Vaccine:
 The vaccines of this invention comprise autologous tumor cells (or an
 alternative source of tumor-associated antigen) and at least one cell
 allogeneic to the host that produces a cytokine of therapeutic importance.
 As described earlier, certain embodiments of this invention comprise a
 plurality of different allogeneic cells, each of which produces a
 different cytokine. Preferably, the cytokines produced by each different
 cell are amongst those listed herein.
 In one method, cell components of the vaccine are prepared and combined in
 bulk at the desired ratio(s) to provide sufficient cells for the entire
 course of treatment envisioned. The mixture is stored frozen, and aliquots
 are thawed seriatim for each administration. This ensures a consistency
 amongst the cell ratio.
 To allow adjustments to components of the vaccine or the ratios used, it is
 generally preferable to assemble the vaccine close to the time of
 administration. Various cell populations may be collected in advance, and
 cultured or cryopreserved as necessary to ensure sufficient numbers of
 cells for administration and testing throughout the planned protocol.
 It is important to remove any additional components used in preparing the
 cells which may have an unwanted effect in the subject. In particular,
 fetal calf serum, bovine serum components, or other biological supplements
 in the culture medium are typically removed so as to avoid an
 immunological side reaction against them. Typically, the cell components
 of the vaccine are washed, such as by repeated gentle centrifugation, into
 a suitable pharmacologically compatible excipient. Compatible excipients
 include isotonic saline, with or without a physiologically compatible
 buffer like phosphate or Hepes and nutrients such as dextrose,
 physiologically compatible ions, or amino acids, and various culture media
 suitable for use with lymphocyte populations, particularly those devoid of
 other immunogenic components. Carrying reagents, such as albumin and blood
 plasma fractions and nonactive thickening agents, may also be used.
 Non-active biological components, to the extent that they are present in
 the pharmacological preparation, are preferably derived from the same
 species, and are even more preferably obtained previously from the subject
 to be treated.
 The vaccine compositions of this invention may optionally include
 additional active components working independently or in concert with the
 tumor associated antigen and activated allogeneic cells. Such optional
 components include but are not limited to isolated or recombinant
 cytokines, particularly those explicitly referred to in this disclosure,
 adjuvants, and other cell types. Preferred additional components are
 bacillus of the M. bovis 15 strain Calmette-Guerin (BCG) or extracts
 thereof, or alternatively, the A60 mycobacterial antigen complex (Maes et
 al.).
 A vaccine composition of this invention is deemed "suitable" for
 administration to a human if reasonable and acceptable standards have been
 taken to ensure that the vaccine itself will not confer additional major
 pathology on the recipient. Side effects such as local inflammation,
 induration, or pain, or a febrile response may be unavoidable and are
 generally acceptable if the treatment is otherwise successful in a
 substantial proportion of patients. However, the composition should be
 reasonably free of: a) unrelated and pathological infectious or chemical
 agents, particularly from the donor of the allogeneic lymphocytes; b)
 undesirable growths as may be generated or propagated in tissue culture,
 such as bacteria or bacterial toxins, mycobacteria, and viruses; c)
 unacceptable levels of oncogenic agents or aggressively growing cancer
 cells not originating from the subject being treated; and d) components
 liable to initiate or effect an undesirable immune reaction, particularly
 anaphylactic shock. Particular tests that can be used are listed in the
 example section of this disclosure.
 The compositions of the present invention, and subcomponents thereof may be
 supplied in unit dosage or kit form. Kits of this invention can comprise
 various components of a cellular vaccine or pharmaceutical composition
 therefor provided in separate containers. The containers may separately
 contain cells or antigens such that when mixed together constitute a
 vaccine of this invention in unit dosage or multiple dosage form.
 Preferred kits comprise in separate containers: cytokine-secreting
 allogeneic cells; and tumor-associated antigen from the human,
 particularly primary tumor cells from the human, or progeny thereof.
 Alternatively, the kits may comprise a cell or cell mixture in one
 container and a pharmaceutical excipient in another container. Preferred
 kits of this nature comprise cytokine-secreting allogeneic cells in one
 container, and an excipient in another. The user can employ the excipient
 to prepare their own tumor antigen or autologous tumor cells, said
 preparation then being combined with the cytokine-secreting cells for
 administration to a subject. Packaged compositions and kits of this
 invention typically include instructions for storage, preparation and
 administration of the composition.
 Use of Cellular Vaccines in Cancer Treatment
 The compositions of this invention may be administered to subjects,
 especially but not limited to human subjects. They are particularly useful
 for eliciting an immune response against a tumor-associated antigen, or
 for treating cancer.
 Objectives of Treatment:
 One purpose of administering the vaccine is to elicit an immune response.
 The immune response may include either humoral or cellular components, or
 both. Humoral immunity may be determined by a standard immunoassay for
 antibody levels in a serum sample from the treated individual.
 Since cellular immunity is thought to play an important role in immune
 surveillance of cancer, generating a cellular immune response is
 frequently a particular objective of treatment. As used herein, a
 "cellular immune response" is a response that involves T cells, and can be
 observed in vitro or in vivo.
 A general cellular immune response may be measured as the T cell
 proliferative activity in cells (particularly PBL) sampled from the
 subject after vaccine administration. Inactivated tumor cells, preferably
 derived from the subject, are used as stimulators. A non-specific mitogen
 such as PHA serves as a positive control; incubation with an unrelated
 stimulator cell serves as a negative control. After incubation of the
 PBMCs with the stimulators for an appropriate period (typically 5 days),
 [.sup.3 H]thymidine incorporation is measured. If desired, determination
 of the subset of T cells that is proliferating can be performed using flow
 cytometry. T cell cytotoxicity (CTh) can also be measured. In this test,
 an enriched T cell population from the subject are used as effectors in a
 standard .sup.51 Cr release assay. Tumor cells are radiolabeled as targets
 with about 200 .mu.Ci of Na.sub.2 .sup.51 CrO.sub.4 for 60 minutes at
 37.degree. C., followed by washing. T cells and target cells
 (.about.1.times.10.sup.4 /well) are then combined at various
 effector-to-target ratios in 96-well, U-bottom plates. The plates are
 centrifuged at 100.times.g for 5 minutes to initiate cell contact, and are
 incubated for 4-16 hours at 37.degree. C. with 5% CO.sub.2. Release of
 .sup.51 Cr is determined in the supernatant, and compared with targets
 incubated in the absence of T cells (negative control) or with 0.1%
 TRITON.TM. X-100 (positive control).
 Another purpose of administering the vaccine is for treatment of a
 neoplastic disease, particularly cancer. Beneficial effect of the vaccine
 will generally be at least in part immune mediated, although an immune
 response need not be positively demonstrated in order for the compositions
 and treatment methods to fall within the scope of this invention, unless
 otherwise required.
 Suitable Subjects:
 The compositions of this invention may be used for administration to both
 human and non-human vertebrates. They provide advantages over previously
 available compositions particularly in outbred populations, and
 particularly in spontaneous tumors. Veterinary applications are
 contemplated within the scope of the invention.
 Cellular vaccines are designed for use in human subjects, and are
 especially suitable for human treatment. The vaccines may be given to any
 human subject with the discretion of the managing physician. Typically,
 the subject will either have cancer, or be at substantial risk of
 developing cancer.
 Typical human subjects for therapy comprise two groups, which may be
 distinguished by clinical criteria. Patients with "advanced disease" or
 "high tumor burden" are those who bear a clinically measurable tumor. A
 clinically measurable tumor is one that can be detected on the basis of
 tumor mass (e.g., by palpation, MRI, CAT scan, X-ray, or
 radioscintigraphy; positive biochemical or histopathological markers on
 their own are insufficient to identify this population).
 A vaccine composition embodied in this invention is administered to
 patients with advanced disease with the objective of palliating their
 condition. Ideally, reduction in tumor mass occurs as a result, but any
 clinical improvement constitutes a benefit. Clinical improvement includes
 decreased risk or rate of progression or reduction in pathological
 consequences of the tumor.
 A second group of suitable subjects is known in the art as the "adjuvant
 group". These are individuals who have had a history of cancer, but have
 been responsive to another mode of therapy. The prior therapy may have
 included (but is not restricted to) surgical resection, radiotherapy,
 traditional chemotherapy, and other modes of immunotherapy. As a result,
 these individuals have no clinically measurable tumor by the definition
 given above. However, they are suspected of being at risk for recurrence
 or progression of the disease, either near the original tumor site, or by
 metastases. The adjuvant group may be further subdivided into high-risk
 and low-risk individuals. The subdivision is made on the basis of features
 observed before or after the initial treatment. These features are known
 in the clinical arts, and are suitably defined for each different cancer.
 Features typical of high risk subgroups are those in which the tumor has
 invaded neighboring tissues, or which show involvement of lymph nodes.
 A vaccine composition embodied in this invention is administered to
 patients in the adjuvant group in order to elicit an anti-cancer response
 primarily as a prophylactic measure against recurrence. Ideally, the
 composition delays recurrence of the cancer, or more preferably, reduces
 the risk of recurrence (i.e., improves the cure rate). Such parameters may
 be determined in comparison with other patient populations and other modes
 of therapy.
 Of course, crossovers between these two patient groups occur, and the
 vaccine compositions of this invention may be administered at any time
 that is appropriate. For example, therapy may be conducted before or
 during traditional therapy of a patient with high tumor burden, and
 continued after the tumor becomes clinically undetectable. Therapy may be
 continued in a patient who initially fell in the adjuvant group, but is
 showing signs of recurrence.
 Examples of tumors that can be treated by the compositions and methods of
 this invention include the following: pancreatic tumors, such as
 pancreatic ductal adenocarinomas; lung tumors, such as small and large
 cell adenocarcinomas, squamous cell carcinoma, and brionchoalveolar
 carcinoma; colon tumors, such as epithelial Eadenocarcinoma and their
 metastases; and liver tumors, such as hepatoma and cholangiocarcinoma.
 Also included are breast tumors, such as ductal and lobular
 adenocarcinoma; gynecologic tumors, such as squamous and adenocarcinoma of
 the uterine cervix, and uterine and ovarian epithelial adenocarcinoma;
 prostate tumors, such as prostatic adenocarcinoma; bladder tumors, such as
 transitional squamous cell carcinoma; tumors of the RES system, such as
 nodular or diffuse B or T cell lymphoma, plasmacytoma, and acute or
 chronic leukemia; skin tumors, such as malignant melanoma; and soft tissue
 tumors, such as soft tissue sarcoma and leiomyosarcoma. Of especial
 interest are brain tumor, such as astrocytoma, oligodendroglioma,
 ependymoma, medulloblastomas, and primitive neural ectodermal tumor.
 Included in this category are gliomas, glioblastomas, and gliosarcomas.
 Also of especial interest is ovarian carcinoma.
 The immune status of the individual may be any of the following: The
 individual may be immunologically naive with respect to certain
 tumor-associated antigens present in the composition, in which case the
 compositions may be given to initiate or promote the maturation of an
 anti-tumor response. The individual may not be currently expressing
 anti-tumor immunity, but can have immunological memory, particularly T
 cell memory relating to a tumor-associated antigen comprised in the
 vaccine, in which case the compositions can be given to stimulate a memory
 response. The individual can also have active immunity (either humoral or
 cellular immunity, or both) to a tumor-associated antigen comprised in the
 vaccine, in which case the compositions may be given to maintain, boost,
 or maturate the response, or recruit other arms of the immune system. The
 subject should be at least partly immunocompetent, so as to minimize a
 graft versus host reaction of pathological scope. However, it is
 recognized that cancer patients often show a degree of immunosuppression,
 and this does not necessarily prevent the use of the compositions of the
 invention, as long as the compositions may be given safely and
 effectively. Immunocompetence in the subject may be of host origin, or may
 be provided by way of a concurrent adoptive transfer treatment.
 Modes of Administration and Dose:
 The compositions of this invention may be administered to the subject at
 any site, particularly a site that is "distal" to or "distant" from the
 primary tumor.
 The route of administration of a pharmaceutical composition may be
 parenteral, intramuscular, subcutaneous, intradermal, intraperitoneal,
 intranasal, intravenous (including via an indwelling catheter), via an
 afferent lymph vessel, or by another route that is suitable in view of the
 tumor being treated and the subject's condition. Because of low-level
 inflammation or induration that may occur for the few days after
 administration, relatively non-invasive methods are preferred,
 particularly subcutaneous routes.
 The dose given is an amount "effective" in bringing about a desired
 therapeutic response, be it the stimulation of an immune response, or the
 treatment of cancer as defined elsewhere in this disclosure. For the
 pharmaceutical compositions of this invention, effective doses typically
 fall within the range of about 10.sup.15 to 10.sup.10 cells, including
 allogeneic cytokine-producing cells, and autologous tumor cells or an
 equivalent thereof. Where a tumor antigen preparation or tumor cell
 extract is used in place of autologous tumor cells, the amount of tumor
 antigen present should be equivalent to what would be provided in the
 level of cells indicated. The number of autologous tumor cells may be
 adjusted to accommodate unusually high or low levels of tumor antigen
 expression. Where a plurality of allogeneic cells genetically altered to
 produce different cytokines is used, the range referred to includes the
 total number of such cells. The number of allogeneic cytokine-producing
 cells is adjusted according to the level of cytokines produced by the cell
 population.
 Preferably, between about 10.sup.6 to 10.sup.9 of allogeneic
 cytokine-producing cells and about 10.sup.6 to 10.sup.9 autologous tumor
 cells are used; more preferably between about 2.times.10.sup.6 and
 5.times.10.sup.8 cells in each cell population is used; more preferably
 between about 5.times.10.sup.6 and 2.times.10.sup.8 cells in each
 population are used; even more preferably between about 1.times.10.sup.7
 and 1.times.10.sup.8 cells in each population are used. Multiple doses,
 when used in combination to achieve a desired effect, each fall within the
 definition of an effective amount.
 The various components of the cellular vaccine are present in an "effective
 combination", which means that there are sufficient amounts of each of the
 components for the vaccine to be effective. This will depend not only on
 the absolute number of cells, but also on the ratio of the various
 components of the vaccine one to another. Preferred ratios of total
 allogeneic cytokine-secreting cells to autologous tumor cells or
 equivalent are 100:1 to 1:100, more typically they are between about 25:1
 and 1:25, even more preferably they are between about 10:1 and 1:10, still
 more preferably they are between about 3:1 and 1:3. Often more important
 than the actual number of cytokine-producing cells used is the
 biosynthetic capability of the cells; fewer cells being required where the
 biosynthetic capability is higher. Preferably, the allogeneic cells in a
 dose of the vaccine are capable of synthesizing at least about 0.1 ng,
 more preferably at least about 0.5 ng, more preferably at least about 2
 ng, even more preferably at least about 10 ng of the cytokine of interest
 during a 1 hour incubation under physiological conditions. Where a
 plurality of different cytokine-producing cells are used, ratios are
 chosen to give appropriate levels of biological activity; typically
 between 25:1 and 1:25, more usually between 5:1 and 1:5 on a molar basis.
 Determination of optimal cell dosage and ratios is a matter of routine
 determination, as described in the example section below, and within the
 skill of a practitioner of ordinary skill, in light of the instructions
 provided herein.
 For embodiments of the invention where the vaccine consists essentially of
 cells autologous to the patient expressing a membrane cytokine, the number
 of cells is between 10.sup.5 and 10.sup.10 per dose; more preferably
 between about 4.times.10.sup.6 and 1.times.10.sup.9 cells per dose; more
 preferably between about 1.times.10.sup.7 and 4.times.10.sup.8 cells per
 dose even more preferably between about 2.times.10.sup.7 and
 2.times.10.sup.8 cells per dose. Multiple doses, when used in combination
 to achieve a desired effect, each fall within the definition of an
 effective amount.
 The pharmaceutical compositions of this invention may be given following,
 preceding, in lieu of, or in combination with, other therapies relating to
 generating an immune response or treating cancer in the subject. For
 example, the subject may previously or concurrently be treated by
 chemotherapy, radiation therapy, and other forms of immunotherapy and
 adoptive transfer. Example 7 describes the use of a vaccine of this
 invention in combination with such chemotherapeutic agents as Cisplatin,
 combination Cisplatin/Cyclophaphamide, Cisplatin/Cyclophosphamide/
 Doxorobicin or Taxol. Where such modalities are used, they are preferably
 employed in a way or at a time that does not interfere with the
 immunogenicity of the compositions of this invention. The subject may also
 have been administered another vaccine or other composition in order to
 stimulate an immune response. Such alternative compositions may include
 tumor antigen vaccines, nucleic acid vaccines encoding tumor antigens,
 anti-idiotype vaccines, and other types of cellular vaccines, including
 cytokine-expressing tumor cell lines.
 In a particular embodiment, the subject will have previously been treated
 with an intra-tumor implant of stimulated allogeneic lymphocytes, such as
 is described in International Patent Application WO 96/29394. Combination
 protocols wherein another mode of vaccination or other therapy preceding
 or following administration of an autologous tumor cell/allogeneic
 cytokine-secreting cell vaccine, are embodied in the present invention.
 Timing of administration is within the judgment of the managing physician,
 and depends on the clinical condition of the patient, the objectives of
 treatment, and concurrent therapies also being administered. At an
 appropriate time in patient management, an initiating dose is given, and
 the patient is monitored for either an immunological or clinical response,
 often both. Suitable means of immunological monitoring include a one-way
 MLR using patient's PBL as responders and primary tumor cells as
 stimulators. An immunological reaction may also be manifest by a delayed
 inflammatory response at the injection site. Suitable means of monitoring
 the tumor are selected depending on the tumor type and characteristics,
 and may include magnetic resonance imaging (MRI), radioscintigraphy with a
 suitable imaging agent, monitoring of circulating tumor marker antigens,
 and the subject's clinical response. An example of an appropriate clinical
 marker is serum CA-125 for the monitoring of advanced ovarian cancer.
 Hempling et al. (1993) J. Surg. Oncol. 54:38-44. Additional doses may be
 given as appropriate, typically on a monthly, semimonthly, or preferably a
 weekly basis, until the desired effect is achieved. Thereafter, and
 particularly when the immunological or clinical benefit appears to
 subside, additional booster or maintenance doses may be given as required.
 When multiple doses of a cellular vaccine are given to the same patient,
 some attention should be paid to the possibility that the allogeneic
 lymphocytes in the vaccine may generate an anti-allotype response. The use
 of a mixture of allogeneic cells from a plurality of donors, and the use
 of different allogeneic cell populations in each dose, are both strategies
 that can help minimize the occurrence of an anti-allotype response.
 During the course of therapy, the subject is evaluated on a regular basis
 for side effects at the injection site, or general side effects such as a
 febrile response. Side effects are managed with appropriate supportive
 clinical care.
 Cell Lines
 This invention includes the cell lines listed in the Table below. These
 cell lines are provided and described for the convenience of the
 practitioner, and may be used, inter alia, for the preparation of certain
 vaccines of this invention, or for methods of treatment of this invention.
 None of the cell lines listed is required for the general practice of the
 invention, except in particular embodiments where a cell line is
 explicitly required.
 TABLE 1
 ATCC
 Designation Origin Description Accession No.
 UCI-107E IL-4 GS Ovarian carcinoma genetically altered to
 cell line UCI-107 express IL-4
 UCI-107M GM-CSF-MPS genetically altered to
 express GM-CSF
 UCI-107A IL-2 AS genetically altered to
 express IL-2
 ACBT Glioblastoma cell line parental
 cell line
 ACBT/TNF-G genetically altered to
 express TNF-.alpha.
 ACBT/IL-4-T genetically altered to
 express IL-4
 ACBT/IL-2-C2 genetically altered to
 express IL-2
 ACBT/GM-CSF-M4 genetically altered to
 express GM-CSF
 Upon allowance and issuance of this application as a United States Patent,
 all restriction on the availability of the deposits will be irrevocably
 removed, and access to the designated deposits will be available during
 pendency of the above-named application to one determined by the
 Commissioner to be entitled thereto, under 37 CFR .sctn. 1.14 and 35 USC
 .sctn. 1.22. Moreover, the designated deposits will be maintained for a
 period of thirty (30) years from the date of deposit, or from five (5)
 years after the last request for the deposit; or for the enforceable life
 of the U.S. patent, whichever is longer.
 The examples presented below are provided as a further guide to a
 practitioner of ordinary skill in the art, and are not meant to be
 limiting in any way.
 EXAMPLES
 Example 1
 An Ovarian Cancer Cell Line Transduced to Express IL-4
 A human ovarian cancer cell line was genetically altered to secrete IL-4,
 using a retroviral vector comprising an IL-4 encoding construct. The cell
 line was stable, and capable of IL-4 biosynthesis even after an
 inactivating dose of radiation. The cell line expressed MHC Class I and
 Her-2/neu antigens, but no MHC Class II antigens, ICAM-1, CA-125, or IL-4
 receptors.
 The human ovarian cell line UCI-107 was established from a previously
 untreated patient with a primary Stage Ill serous papillary adenocarcinoma
 of the ovary. The UCI-101 and UCI-107 cell lines have been previously
 characterized (Gambea-Vujicic et al. Submitted, Gynecol. Oncol.) and were
 kindly provided by Dr. Alberto Manetta (University of California, Irvine
 Medical Center). Cells were maintained at 37.degree. C., 5% CO.sub.2 in
 complete media (CM) containing RPMI 1640 (Gibco Life Technologies), 10
 percent fetal bovine serum (FBS, Gemini Bioproducts, Calabassas, Calif.),
 and 1 percent penicillin/streptomycin sulfate (Irvine Scientific, Santa
 Ana, Calif.).
 Retroviral vectors were constructed as follows: The pLXSN plasmid was
 kindly provided by Dr. A. Dusty Miller (Fred Hutchinson Cancer Center,
 Seattle, Was.). This plasmid, derived from a Maloney murine leukemia virus
 (MLV) contains the neophosphotransferase gene whose constitutive
 expression is driven by the SV40 enhancer/promoter, the 5' retroviral LTR
 of the integrated vector drives the expression of an inserted gene. The
 human IL-4 cDNA was obtained from ATCC in the Okayama and Berg pCD cloning
 vector, and was excised using BamHI restriction enzyme. Okayama et al.
 (1983) Mol. Cell. Biol. 3:228-289. The cDNA was then cloned into the BamHI
 restriction site in the multiple cloning region of pLSXN. Proper
 orientation of the cDNA was determined by diagnostic restriction
 endonuclease digests. Once constructed, retroviral plasmid DNA was then
 purified by CsCl gradient density centrifugation.
 Purified retroviral plasmid DNA (LXSN/IL-4) was used to transduce the
 murine esotropic packaging cell line GP-E86 by the calcium phosphate
 method. Forty-eight-hour supernatant from these cells was then used to
 infect the murine amphotropic-packaging cell line, 17. The
 PA-317-packaging cell line was obtained from the ATCC and maintained in
 CM. Transduced 17 cells were selected by resistance to G418. Isolated
 clones were A expanded, aliquoted, and frozen under liquid nitrogen in a
 master cell bank. The supernatant from a transduced 17 clone,
 containing infectious, replication-incompetent retrovirus, was used to
 infect the human carcinoma cell lines. Briefly, human ovarian carcinoma
 cell lines were seeded in 100-mm tissue culture dishes at densities of
 1.times.10.sup.6 cells in 10 ml CM and incubated for 4 hr at 37.degree.
 C., 5% CO.sub.2 to allow adherence. After incubation, the medium was
 aspirated and replaced with 5 ml of 2% polybrene in phosphate-buffered
 saline (PBS), (Aldrich Chemical Co. Inc., Milwaukee, Wis.). After 30 min
 at 37.degree. C., 5% CO.sub.2, 10 ml of retroviral supernatant was added,
 and retroviral-mediated gene transfer was accomplished by overnight
 incubation. Supernatants were then aspirated and replaced with CM. After
 an additional 48-hr incubation in CM at 37.degree. C., 5% CO.sub.2,
 selection of transduced clones was accomplished by culture in CM
 containing 0.075% G418 (geneticin, Gibco Life Technologies). Clones were
 isolated after 14 days using sterile 8.times.8 8-mm cloning cylinders
 (Belco Glass, Inc., Vineland, N.J.) and expanded for 3 weeks in CM
 containing G418. Parent cell lines were used as positive controls for G418
 resistance. After clonal selection in G418, transduced cell lines were
 returned to CM for expansion and study.
 Cells were established in CM at a density of 0.5.times.10.sup.6 cells/10 ml
 in 100 mm tissue culture dishes. Cell counts were conducted every 12, 24,
 48, 72 and 96 hours, and the number of viable cells was determined using
 trypan blue exclusion. Experiments were conducted to compare the growth of
 non-transduced (parental) and transduced tumor cell lines and to evaluate
 the level of cytokine production over time. Supernatants were collected
 and frozen at -20.degree. C. (for subsequent ELISA evaluation of cytokine
 levels) and culture dishes trypsinized to determine cell count and
 viability.
 Parental, IL-4 transductants, and vector control cells, were seeded in 100
 mm tissue culture dishes (Corning) at a density of 1.times.10.sup.6
 cells/ml in 10 ml CM. After 48 hour incubation at 37.degree. C., 5 percent
 CO.sub.2, supernatant was aspirated, rendered cell-free by centrifugation
 at 1,500 rpm for 10 minutes, then stored at -20.degree. C. IL-4
 concentration was then determined by ELISA, employing a commercially
 available kit (Research & Diagnostic Systems, Minneapolis, Minn.). Table 2
 shows the level of secretion of Interleukin-4 from clones of genetically
 altered human serous papillary ovarian cancer cells
 TABLE 2
 Transduced UCI-101 clones Transduced UCI-107 clones
 Designation IL-4 pg/mL Designation IL-4 pg/mL
 A 140 A 32
 B (not detectable) B 83
 C 49 C 90
 D 40 D 35
 E 87 E 1300
 G 93 F 30
 H 38 G 80
 I 93 H 513
 L 42 L 170
 M 32 M 297
 N (not detectable) N 265
 O (not detectable) P 330
 Q 615
 X 79
 Y 68
 Average 51.1 Average 265.8
 As expected, each parental line and cells transduced with vector alone did
 not produce detectable levels of IL-4. The best IL-4 producing clone,
 termed UCI 107E IL-4 GS, was expanded and employed to from a master cell
 bank for further testing and extensive characterization.
 The parental cell line UCI 107 has the characteristic morphology of ovarian
 epithelial cells grown in vitro. The morphology of UCI 107 cells
 transduced with the LXSN vector alone or LXSN containing the IL-4 gene was
 indistinguishable from that of parental 107 cells. The doubling time of
 parental, vector control, and UCI 107E IL-4 GS cells was determined to be
 15.3, 15.7, and 18.6 hr, respectively.
 FIG. 1 shows the growth and IL-4 secretion by UCI 107E IL-4 GS cells. No
 changes in the growth rate of these cells have been observed in vitro over
 35 passages and 6 months of culture. Levels of IL-4 production were
 consistently in the range of 900 to 1300 pg/ml/10.sup.5 cells/48 hr during
 the 6 months of passage. Extensive tests performed on the UCI 107E IL-4 GS
 master cell bank (MCB) revealed that this line is free of the presence of
 mycoplasma, bacteria, and infectious viruses.
 Southern analysis was conducted using the Neo.sup.R gene to probe the UCI
 107E IL-4 and the parental UCI 107 line. Briefly, concentrated suspensions
 of tissue culture cells were lysed in TNE buffer (10 mM Tris, 100 mM NaCl,
 1 mM EDTA, pH 7.5) containing 0.5% SDS, treated with 50 .mu.g/ml
 proteinase K overnight at 37.degree. C., then extracted with phenol and
 chloroform. The DNA solution was precipitated in 100% ethanol, spooled out
 and resuspended in 10 mM Tris, 0.1 mM EDTA (pH 8). Ten .mu.g of high
 molecular weight DNA was digested with SstI (GIBCO/BRL, Grand Island,
 N.Y.), separated by electrophoresis on a 0.8% agarose gel and transferred
 to Gene Screen Plus (Dupont NEN, Boston, Mass.). Transfer, hybridization,
 and washing were performed according to manufacturer's specifications.
 Random primer IL-4 probe was prepared by the method of Tabor and Struhl
 (1988) In Current Protocols in Mol. Biol. vol. 1:pp 2.2.1-2.2.3. The
 results confirmed that after 20 passages, UCI 107E IL-4 still contained
 the vector DNA.
 Stability of IL-4 secretion to irradiation was tested as follows: Cells
 were irradiated in a 15 ml conical tube in CM at room temperature with
 gamma rays (Cesium 137) at a dose rate of 200 rads/minute. Immediately
 after irradiation, cells were seeded in a Petri dish culture plate at a
 density of 1.times.10.sup.6 cells in 10 ml of CM. Test doses of 1,000 to
 10,000 rads were applied. Irradiated cells were cultured at 37.degree. C.
 in a 5% CO.sub.2 atmosphere and the medium was completely changed every
 four days in all the dishes. Every 48 hours, culture supernatants were
 collected from the dish for cytokine production and the number of viable
 cells was assessed by light microscopy by trypan blue exclusion.
 Results of this experiment are shown in FIGS. 2A-C. Cells irradiated with
 between 2,500 and 10,000 rads remained viable for about 8 days but all the
 cells were dead by 3 weeks. Cells irradiated with 1,000 rads recuperated
 and continued to proliferate. Levels of cytokine production were
 detectable for 8 days at all doses and closely paralleled the number of
 viable cells. FIG. 2B shows IL-4 production after irradiation at 5,000
 rads (.quadrature.) or 10,000 rads (.box-solid.) in three separate
 experiments. FIG. 2C shows IL-4 production standardized in pg/ml/10.sup.5
 cells/48 hr by UCI 107E IL-4 GS cells after irradiation at 5,000 or 10,000
 rads in two separate experiments. No statistically significant differences
 in survival were seen among cells irradiated with 2,500, 5,000, and 10,000
 rads on days 2 (p=0.72), 4 (p=0.14), 6 (p=0.10), and 8 (p=0.3).
 Proteins of the major histocompatibility complex, adhesion molecules, and
 tumor-associated antigens such as CA-125 are important for both
 recognition and destruction of tumor cells by the immune system.
 Accordingly, expression of representative antigens was examined by
 fluorescence-activated cell sorting (FACS). Monolayers of parental cells,
 vector controls, and IL-4 transduced cells were harvested with 0.1%
 trypsin and 0.2% EDTA. Harvested cells were fluorescently labeled using
 the following primary antibodies: anti-HLA class I and anti-HLA class II
 (monoclonal antibodies (mAb) W6/32 and CR3-43, respectively; Accurate
 Chemical and Scientific Corp.), anti-ICAM-1 (mAb LB-2; Becton-Dickinson);
 anti-CA-125 (mAb OC125; Signet Laboratories); anti-HER-2/new p185 (mAb
 TA-1; Oncogene Science), and anti-IL-4-receptor (Genzyme Diagnostic).
 The expression of surface antigens detected by FACS analysis is illustrated
 in FIGS. 3A-C. Parental cells, vector controls, and 107E IL-4 GS cells
 constitutively express MHC class I antigens and Her-2/neu, but did not
 express MHC class II antigens, CA-125, ICAM-1, or IL-4 receptors.
 Expression of surface antigens was also determined at 2 or 8 days after
 irradiation. MHC class I antigen and Her-2/new antigen expression
 increased significantly at all radiation doses, and tended towards higher
 expression at higher doses. Irradiation did not induce expression of HLA
 class II antigens, ICAM-I, or CA-125.
 Collectively, these results indicate that UCI 107E IL-4 GS cells constitute
 a stable IL-4 secreting cell line. The cells can be irradiated to stop
 replication effectively, yet maintain IL-4 production for up to a week.
 Example 2
 An Ovarian Cancer Cell Line Transduced to Express GM-CSF
 A human ovarian carcinoma cell line (UCI-107) was genetically engineered to
 secrete human cytokine granulocyte-macrophage colony stimulating factor
 (GM-CSF), similar to the method described in Example 1. One clone, termed
 UCI-107M GM-CSF-MPS, constitutively secretes high levels of GM-CSF
 (.about.500 pg/ml/105 cells 48 hours) UCI-107M GM-CSF-MPS cells express
 MHC Class I and Her2/New surface antigens, but do not express detectable
 MHC Class II, ICAM-I or the tumor-associated antigen CA-125. After a
 radiation dose of 10,000 rads, GM-CSF secretion continued until about Day
 8.
 The choice of transducing an ovarian carcinoma cell line with the GM-CSF
 gene has been made in light of the important role of GM-CSF in the
 maturation and function of specialized antigen-presenting cells. GM-CSF is
 one of the most potent stimulators of systemic anti-tumor immunity.
 The pLXSN plasmid is described in Example 1. The human GM-CSF cDNA was
 obtained from the ATCC in the Okayama and Berg pCD cloning vector, and was
 excised using BamHI restriction enzyme. The cDNA was then cloned into the
 BamHI restriction site in the multiple cloning region of pLSXN. Proper
 orientation of the cDNA was determined by diagnostic restriction
 endonuclease digests. Once constructed, retroviral plasmid DNA was then
 purified by CsCl gradient density centrifugation.
 Purified retroviral plasmid DNA (LXSN/GM-CSF) was used to transfect the
 murine esotropic packaging cell line GP-E86 as before. Forty-eight hour
 supernatant from these cells was then used to infect the murine
 amphotropic packaging cell line 17, and selected by resistance to G418.
 The supernatant from a transduced 17 clone, containing infectious,
 replication incompetent retrovirus, was used to infect the UCI-107 cell
 line. After clonal selection in G418, transduced cell lines were returned
 to CM for expansion and study.
 Parental, GM-CSF transducts and vector control cells, were seeded in 100 mm
 tissue culture dishes (Corning) at a density of 1 x 10.sup.6 cells/ml in
 10 ml CM. After 48 h incubation at 37.degree. C., 5% CO.sub.2, supernatant
 was aspirated, centrifuged at 1,500 rpm for 10 minutes, then stored at
 -20.degree. C. GM-CSF concentration was determined by ELISA, employing a
 commercially available kit (Research & Diagnostic Systems, Minneapolis,
 Minn.). The biologic activity of GM-CSF was measured in a cell
 proliferation assay using a GM-CSF factor-dependent human cell line, TF-1,
 provided by Dr. Monica Tsang (Research and Diagnostic Systems). The level
 of biologic activity correlated with the level of GM-CSF detected by
 ELISA.
 Cultures of each transduced LXSN-GM-CSF clone and LXSN vector control were
 established for 48 h, and the media tested for the presence of GM-CSF. As
 expected, parental UCI-107 cells and cells transduced with the LXSN vector
 alone did not produce detectable levels of GM-CSF.
 TABLE 3
 Transduced UCI-107 clones
 Designation GM-CSF pg/mL Designation GM-CSF pg/mL
 A 55 L 126
 A1 15 L1 149
 B 25 M 420
 B1 98 M1 67
 C 9 N 31
 C1 7 N1 63
 D 83 O 79
 D1 73 O1 115
 E 7 P 31
 E1 61 Q 49
 F 5 R 35
 F1 21 S not detectable
 G 47 T 74
 G1 39 U 34
 H 13 V not detectable
 H1 134 X 8
 I 86 Y 146
 I1 12 Z 22
 Of 36 clones originally selected, the highest GM-CSF producing clone,
 termed UCI-107M GM-CSF-MPS, was expanded and employed to form a master
 cell bank for further testing and extensive characterization:
 The parental cell line UCI 107 has the characteristic morphology of ovarian
 epithelial cells grown in vitro. The morphology of UCI 107 cells
 transduced with the LXSN vector alone or LXSN containing the GM-CSF gene
 was indistinguishable from that of parental 107 cells. The doubling time
 of the parental, vector control and UCI-107M GM-CSF-MPS cells was
 approximately 20 to 26 h.
 Over a period of 6 months and a total of 35 passages, levels of GM-CSF
 production by UCI-107M GM-CSF-MPS were consistently in the range of 420 to
 585 pg/ml/10.sup.5 cells/48 hours. The GM-CSF secreting clone was
 evaluated for successful gene insertion by Southern hybridization after 20
 passages of the cells, and the presence of the inserted gene was
 confirmed.
 To determine the stability of GM-CSF secretion after irradiation, UCI-107M
 GM-CSF-MPS cells were irradiated and supernatants from individual
 subcultures were evaluated for cytokine production No effects on cell
 growth were observed at doses of less than 1,000 rads. At higher doses,
 approximately 90% of the cells were viable 48 h after irradiation, with
 30% and 10% viability at 4 and 6 days, respectively. All cells were dead
 after three weeks. No statistically significant differences in survival
 were seen among cells irradiated with 2,500, 5,000 or 10,000 rads.
 Secretion of GM-CSF continued until about day 8, suggesting a decrease but
 not a complete inhibition in the biosynthesis and release of the cytokine
 in cells surviving irradiation.
 The expression of membrane antigens was examined by FACS analysis on
 parental, LXSN vector control and UCI-107M GM-CSF-MPS cells. All three
 cells constitutively express MHC Class I antigens (parental cells: 87.5%,
 non-fluorescence index (MFI)=33.6; GM-CSF secreting cells: 92-9%,
 MFI=53.9); Her-2/neu, (95.8%, MFI=20.8; and 93.3%, MFI=22.1%
 respectively), but did not express MHC Class II determinants, CA-125 or
 ICAM-1. The production of GM-CSF or the presence of the LXSN vector had no
 detectable effect on the expression of these antigens. Increased
 expression of MHC Class I and Her-2/neu surface antigens was observed in
 irradiated cells compared with the non-irradiated controls. Gamma
 irradiation did not induce neo-expression of antigens not originally
 expressed on the parental cells; in particular, BLA Class II antigens,
 ICAM-I or CA-125.
 Example 3
 An Ovarian Cancer Cell Line Transduced to Express IL-2
 A human ovarian carcinoma cell line (UCI-107) was genetically engineered to
 secrete the cytokine Interleukin-2 (IL-2), by retroviral mediated gene
 transduction similar to the method outlined in Example 1. This line was
 transduced with the LXSN retroviral vector containing the human IL-2 gene
 and the neomycin resistance selection marker. One clone termed UCI-107A
 IL-2 AS, was shown to constitutively secrete high levels of IL-2 (i.e.,
 2,000 to 2,300 pg/ml/10.sup.5 cells/48 hours) for over 35 passages and six
 months of study. Unlike parental and vector transduced cells (both of
 which were diploid), UCI-107A IL-2 AS failed to express MHC Class I and
 Her2/Neu surface antigens. In addition, UCI-107A IL-2 AS cells exhibited a
 distinct in vitro morphology, and were resistant to gamma irradiation.
 The human IL-2 cDNA was obtained from ATCC in the Okayama and Berg pCD
 cloning vector and was excised using BamHI restriction enzyme. The cDNA
 was then cloned into the BamHI restriction site in the multiple cloning
 region of the pLSXN plasmid. Proper orientation of the cDNA was determined
 by diagnostic restriction endonuclease digests. Retroviral plasmid DNA was
 purified by CsCl gradient density centrifugation.
 The purified retroviral plasmid DNA (LXSN/IL-2) was used to transfect the
 murine esotropic packaging cell line GP-E86 by the calcium phosphate
 method. Forty-eight hour supernatant from these cells was then used to
 infect the murine amphotropic packaging cell line, 17 obtained from
 ATCC, and selected by resistance to Geneticin. UCI-107 cells were seeded
 into 100 mm tissue culture dishes at densities of 1.times.10.sup.6 cells
 in 10 ml CM. 10 ml of retroviral supernatant was added, and incubated with
 the cells overnight Clones were isolated after 14 days and expanded for
 three weeks in CM containing G418.
 IL-2 concentration in clone supernatants was determined by ELISA, employing
 a commercially available kit (Research & Diagnostic Systems, Minneapolis,
 Minn.). The biologic activity of IL-2 was measured in a cell proliferation
 assay using an IL-2 dependent murine cytotoxic cell line, CTLL-2 provided
 by Dr. Monica Tsang (Research and Diagnostic Systems). The level of
 biologic activity correlated with the level of IL-2 detected by ELISA.
 As expected, parental UCI-107 cells and cells transduced with the LXSN
 vector alone did not produce detectable levels of IL-2. Of 13 clones
 selected, the highest IL-2 producer, termed UCI-107A IL-2 AS, was shown to
 have a level of IL-2 production consistently in the range of 2,000 to
 2,300 pg/ml/10.sup.5 cells/48 hours during the six months of the
 observation period. This clone was expanded and employed to form a master
 cell bank for further testing and characterization.
 The morphology of UCI-107 cells transduced with the LXSN vector was
 indistinguishable from that of parental UCI-107 cells. In contrast,
 UCI-107 cells containing the IL-2 gene exhibited significantly altered
 morphology, being much more spindle-shaped than parental cells. The
 doubling time of parental, vector control and UCI-107A IL-2AS cells was
 approximately 20 to 26 hours, stable for over 35 passages and 6 months of
 culture.
 The IL-2 secreting clone transduced with LXSN-IL-2 and selected in G418 was
 evaluated for successful gene insertion by Southern hybridization probing
 for the Neo.sup.R gene confirmed the presence of the retroviral vector in
 the genome of the transduced UCI-107 cells.
 In irradiation tests, no effects on cell growth were observed at doses of
 less than 5,000 rads. At doses of 10,000 rads, approximately 90 percent of
 the cells were viable 48 hours after irradiation, 45 percent after 4 days
 and 10 percent after 6 days. All the cells were dead after three weeks.
 IL-2 production was maintained at high levels for about 6 days, after
 which cytokine secretion rapidly decreased parallel with the viable cell
 number.
 Parental UCI-107 cells and vector control cells express MHC Class I
 antigens, 87.5% and 94.8% respectively; Her-2/neu, 95.8% and 94.3%
 respectively; but did not express MHC Class II determinants, CA 125 or
 ICAM-1. In contrast, UCI-107A IL-2 AS cells did not display any of these
 antigens at detectable levels.
 Seven and eight, 7 week-old female BALB/C nude mice, respectively, were
 injected i.p. with 10.times.10.sup.6 LXSN vector control or UCI-107A IL-2
 AS cells, and the animals were followed for eight weeks to evaluate tumor
 formation. Vector control cells formed large solid tumor nodules within 3
 weeks after injection, and all the animals died within 25 days. In
 contrast, seven of eight nude mice injected with UCI-107A IL-2 AS cells
 remained alive and tumor-free after eight weeks.
 Susceptibility of tumors to lysis by freshly isolated PBL from normal
 donors was tested in 4- and 18-hour .sup.51 Cr release cytotoxicity assays
 and long-term cytotoxicity assays (Finke et al.). Cytotoxic activity was
 observed against UCI-107 parental and vector control cells at four hours.
 In contrast, the UCI-107A IL-2 AS cells were killed only after 18 h or
 longer:
 TABLE 4
 Effector 4 hr .sup.51 Cr 18 hr .sup.51 Cr 72 hr
 Long
 to Target Ratio Release Release Term
 Target Cell Employed (E:T) Assay Assay Killing Assay
 UCI 107 25:1 28.0 .+-. 1.1* 42.3 .+-. 2 44.5 .+-.
 2.5
 ENTAL 10:1 2.0 .+-. 1.6 13.7 .+-. 3.1 11.0 .+-. 0
 5:1 2.7 .+-. 1.2 2.7 .+-. 1.2 9.0 .+-. 2.0
 2.5:1 0 .+-. 0 0 .+-. 0 not done
 UCI 107 25:1 29.9 .+-. 2.0 42.7 .+-. 0.9 48.5 .+-.
 5.5
 LXSN 10:1 6.2 .+-. 1.0 10.8 .+-. 6.7 18.0 .+-.
 6.5
 (vector control) 5:1 2.5 .+-. 0.6 1.6 .+-. 1.6 13.0 .+-. 6.0
 2.5:1 0 .+-. 0 0 .+-. 0 not done
 UCI 107A 25:1 0 .+-. 0 54.4 .+-. 10.4 76.0 .+-. 0
 IL-2 AS 10:1 0 .+-. 0 28.4 .+-. 1.6 75.0 .+-. 0
 5:1 0 .+-. 0 2.4 .+-. 2.4 69.5 .+-. 0.5
 2.5:1 0 .+-. 0 0 .+-. 0 not done
 *Mean .+-. SD
 The resistance of the UCI-107A IL-2 AS cell to cytotoxic killing in these
 in vitro assays may provide an advantage to the use of these cells in
 vaccine compositions. The lack of expression of MHC Class 1, Class II and
 ICAM-1 antigens may reduce the anti-allogeneic response that would
 ordinarily eliminate cells of the vaccine.
 Example 4
 Cytokine-Expressing Human Glioblastoma Cells
 In this example, human glioblastoma cells were genetically altered to
 secrete TNF-.alpha., IL-2, IL-4, or GM-CSF. The engineered cell lines were
 characterized for potential use in vaccine trials.
 A human glioblastoma cell line designated ACBT was established as follows:
 Tumor tissue was obtained at the time of surgery from a 42 year old male
 patient with recurrent glioblastoma multiforma of the right temporal lobe.
 Tumor tissue was minced into 20 mm pieces and digested overnight in
 collagenase (Sigma Chemical Co., N.J.). The cell suspension was then
 filtered through a sterile 100 pm nylon mesh (Tetro Inc.), and washed 3
 times in RPMI 1640 (GIBCO Life Technologies, Grand Island, N.Y.), 10%
 fetal bovine serum (FBS; GIBCO Life Technologies) and 1%
 penicillin/streptomycin. Cells cultured from the original tissue were
 confirmed to be glioblastoma in origin as determined by GFAP positively
 and morphology.
 The cDNA gene for human TNF-ct was obtained from the ATCC. It was cloned
 into the Hpal restriction site in the multiple cloning region of plasmid
 pLNCX (Example 1) by a double blunt end ligation to generate the
 recombinant plasmid pLNCT. Proper sense orientation of the TNF cDNA was
 confirmed by analytical restriction enzyme digests. The expression of
 TNF-.alpha. in the LNCT vector is controlled by the internal immediate
 early enhancer/promoter of the cytomegalovirus, and the expression of the
 neomycin resistance gene is under control of the 5' Long Terminal Repeat
 (LTR) of the integrated retroviral vector.
 The IL-2, IL-4 and GM-CSF cDNA containing vectors were constructed in a
 similar fashion. The cDNA for each of these cytokines was obtained from
 ATCC and was cloned into the BamHI restriction site in the multiple
 cloning region of plasmid pLXSN to generate the recombinant plasmids
 pLXSN/IL-2, pLXSN/IL-4, and pLXSN/GM-CSF. Cytokine expression in this
 vector system is controlled by the 5' LTR of the integrated proviral
 vector, and expression of the neomycin resistance gene is under control of
 the SV40 early promoter.
 Infectious TNF gene containing retroviral particles were generated by
 transfecting the amphotropic packaging cell line 17 with purified pLNCT
 DNA, or with pLNCX DNA (the vector control). In brief, the procedure
 comprised combining 10 Ag of CsCl purified plasmid of pLNCT DNA with 10 g
 calf thymus DNA. The DNA mixture was used to transfect 1.times.10.sup.6
 17 cells, which were seeded 4 hours prior in a 100 mm tissue culture
 dish, by the calcium phosphate precipitation method. After a 48-hour
 incubation period, the media was removed from the transfected cells and
 centrifuged at 3,000.times.g for 5 minutes to remove cellular debris. The
 resulting supernatant contained infectious retroviral particles (vLNCf)
 and was used to transduce ACBT cells. The transfection process comprised
 plating 1.times.10.sup.4, 1.times.10.sup.5, 10.times.10.sup.6 and
 2.times.10.sup.6 ACBT cells in 10 ml of media in 100-mm tissue culture
 dishes at 37.degree. C. in a 5% CO.sub.2 incubator to allow for cells to
 adhere. After incubation, the media was replaced with 5 ml of polybrene
 (20 .mu.g/ml in phosphate buffered saline, pH 7.4, PBS), and incubated for
 hour at 37.degree. C. The polybrene was removed, and the 17 viral
 containing supernatant was added to the ACBT glioblastoma cells. After 24
 hours, the viral supernatant was replaced by 10 ml. of selection media
 containing G418 (733 .mu.g/ml active G418) at a concentration of 600
 .mu.g/ml. Individual colonies of G418 resistant ACBT/TNF-.alpha. clones
 were isolated after 14 days using sterile 8.times.8 mm glass cloning
 rings, and expanded in CM containing G418 for several weeks. The
 transfected cell lines were then returned to CM for maintenance and
 expansion to create vector control clones of which ACBT/LNCS was isolated
 and used as a vector control cell line.
 Gene transfer using the IL-2, IL-4 and GM-CSF containing vectors were
 conducted in similar fashion to that of TNF-.alpha.. Recombinant DNA
 plasmid containing these cytokine genes were used first to transfect the
 esotropic packaging cell line GP+E-86. After a 48 hour incubation period,
 the media was removed and centrifuged at 3,000.times.g for 5 minutes. The
 viral particle containing supernatant was then used to infect the
 amphotropic packaging cell line 17. Afterwards, individual 17 clones
 were selected by their ability to grow in culture media containing G418.
 The supernatants from individual 17 clones secreting infectious
 LXSN/IL-2, LXSN/IL-4 or LXSN/GM-CSF viral particles were used to transduce
 ACBT glioblastoma cells in a similar fashion to the LNCT transfection as
 described above.
 TNF secretion was determined by ELISA using a polyclonal antibody against
 recombinant human TNF. IL-2, IL-4 and GM-CSF secretion by each of the
 respective transduced ACBT clones were determined by ELISA, employing
 commercially available kits. Results are shown in Tables 5 and 6:
 TABLE 5
 TNF IL-2 IL-4 GM-CSF
 pg/ml pg/ml pg/ml
 ACBT 24 212 12 22
 parental line
 LNCX 24 -- --
 (vector control for TNF)
 LXSN -- 28 12 16
 (vector control for IL-2,
 IL-4, & GM-CSF)
 TABLE 5
 TNF IL-2 IL-4 GM-CSF
 pg/ml pg/ml pg/ml
 ACBT 24 212 12 22
 parental line
 LNCX 24 -- --
 (vector control for TNF)
 LXSN -- 28 12 16
 (vector control for IL-2,
 IL-4, & GM-CSF)
 The ACBT/parental and vector control cells produced minimal to low levels
 of each of the respective cytokines. Clones ACBT/TNF-G, ACBT/IL-2-C2,
 ACBT/IL-4-T and ACBT/GM-CSF-M4 produced the highest levels of the
 indicated cytokine and were used for analysis of growth characteristics
 and kinetics of cytokine secretion.
 The parental cell line, ACBT, has a characteristic morphology of spindle
 shaped astrocytoma cells grown in vitro. The ACBT cells transduced with
 pLXSN or pLNCS vectors alone, or with the vectors containing the cytokine
 genes, exhibited morphologies similar to that of the parental cell. The
 growth rates of parental, LNCS vector control, LXSN vector control,
 ACBT/IL-2-C2, ACBT/IL-4T and ACBT/GM-CSF-M4 cells were similar, with
 doubling times ranging from 25 to 35 hours. The ACBT/TNF-G cell line had a
 somewhat slower growth rate of 41 hours.
 FIG. 4 shows cytokine production by 0.5.times.10.sup.6 cells/10 ml over a
 period of 96 hours. Upper left, clone ACBT/TNF-G; lower left, clone
 ACBT/IL-4-T; upper right, clone ACBT/IL-2-C2; lower right, clone
 ACBT/GM-CSF-M4. Cytokine production increases steadily over time as a
 result of cell replication. The best TNF-producing clone, ACBT/TNF-G, was
 expanded to form a master cell bank for further testing and extensive
 characterization. TNF production was studied over a period of 2.5 months
 and 15 passages, and was consistently in the range of 2500 to 3500 pg/ml.
 Southern blot analysis of DNA from the ACBT/TNF-G cell line confirms that
 the TNF-.alpha. encoding vector is integrated into the genome of the host
 cell.
 ACBT/TNF-G cells were given either 10,000 (.quadrature.) or 20,000
 (.box-solid.) rads of .gamma.-irradiation from a cesium source, and then
 were established as monolayer cultures in CM (FIG. 5). 59% and 45% of the
 cells were viable 48 hours after irradiation with 10,000 and 20,000 rads,
 respectively; and only 2% of cells were viable 14 days later in each
 group. At 3 weeks, all of the cells that had received 20,000 rads were
 dead. Some cells that had received 10,000 rads survived, and replicated to
 44% of the original cell number plated (Panel A). TNF production in the
 culture initially decreased after irradiation, but then steadily increased
 up to day 10 (Panel B). TNF production expressed per 10.sup.5 viable cells
 steadily increased over time after irradiation. There was no observable
 difference in the levels of cytokine secretion per 10.sup.5 viable cells
 between the treatment groups.
 The expression of MHC antigens was analyzed by FACS on parental, vector
 control and ACBT/TNF-G cells. The results obtained are summarized in Table
 7.
 TABLE 7
 Isotype MHC Class I MHC Class II CD54 (ICAM-I)
 Cells MCF % pos MCF % pos MCF % pos MCF % pos
 ACBT 147.6 (6.9%) 859.9 (62.6%) 136.0 (5.7%) 277.6 (39.6)
 parental cells
 ACBT 84.1 (3.3%) 606.3 (50.8%) 156.6 (3.3%) 239.4 (28.9%)
 LNCX vector
 control
 ACBT/TNF-G 24.3 (5.1%) 643.9 (99.9%) 22.5 (4.4%) 225.9 (97.5%)
 transduced
 unirradiated
 ACBT/TNF-G 40.9 (8.4%) 878.1 (99.9%) 34.7 (5.0%) 392.5 (97.8%)
 2 d after
 10,000 rads
 ACBT/TNF-G 34.4 (4.9%) 828.2 (99.9%) 36.6 (4.8%) 327.9 (97.9%)
 2 d after
 20,000 rads
 Parental, LNCX vector control and ACBT/TNF-G cells and expressed MHC Class
 I and CD54 (ICAM-1) surface antigens. The mean channel fluorescence (MCF)
 of cells expressing these antigens were similar for the parental, vector
 control and ACBT/TNF-G cell lines. However, the percent positive cells
 (shown in parentheses) was greater for the TNF-producing clone. ACBT
 parental cells did not express Class II antigens, and transduction with
 the TNF gene did not cause neo-expression of Class II antigens.
 .gamma.-irradiation mildly upregulated the expression of both Class I and
 ICAM-1 surface antigens on ACBT/TNF-G cells at both 10,000 and 20,000 rads
 with increases in the MCF. .gamma.-irradiation did not, however, cause
 neo-expression of Class II molecules on the transduced cell line.
 The ACBT/TNF-G cell line was extensively tested for the presence of various
 microorganisms by our own and outside laboratories. The results revealed
 that the ACBT/TNF-G cell line is free of mycoplasma, bacteria, the DNA
 viruses Epstein-Barr, human hepatitis B, human cytomegalovirus, and
 replication competent retroviruses.
 Example 5
 Membrane M-CSF Expression in a Syngeneic Vaccine Model
 Expression of the M-CSF gene results in two different isoforms of the M-CSF
 protein due to alternative post-transcriptional splicing within exon 6.
 One form of the 5 protein is secreted as a 45 kd homodimeric glycoprotein,
 and the other form remains associated with the cell membrane. Stein et al.
 (1991) Oncogene 6:601. The secreted form (sM-CSF) induces proliferation
 and differentiation of monocyte progenitors, is responsible for the
 stimulation of the effector functions of macrophages such as cytokine
 production and enhanced tumoricidal activity, and may function as a
 chemoattractant for circulating monocytes. In the brain, sM-CSF is
 believed to induce proliferation and activation of microbial cells.
 Alterman et al. (1994) Alt. Chem. Neuropathol. 21:177. The membrane bound
 isoform (mM-CSF) has also been shown to be biologically functional in that
 it is capable of stimulating macrophage colony formation of bone marrow
 stem cells. Stein et al. (1990) Blood 76:1308. T9 glioblostoma cells
 expressing the mM-CSF isoform, but not the secreted form, are killed in
 vitro by tumoricidal macrophages.
 In this example, the therapeutic value of the two M-CSF isoforms was
 investigated in glioma destruction by using T9 glioblastoma cells
 genetically modified to express either the soluble or the
 membrane-associated isoforms of M-CSF in a syngeneic Fischer rat brain
 tumor model.
 T9 glioblastoma tumor cells (induced by the repeated intravenous injection
 of N-nitrosomethylurea in a Fischer F344 rat; Benda et al. (1971) J.
 Neurosurg. 34:310 were provided by Dr. J. Yoshida, Department of
 Neurosurgery, Nagoya University, Japan. An intracranial (i.c.) injection
 of 1.times.10.sup.5 T9 cells into the Fischer rat brain is 100% lethal in
 20-25 days. 9L gliosarcoma cells (induced by repeated i.v. injection of
 N-nitrosomethylurea; 25 Albright et al.) were obtained from Dr. Carol
 Kruse, University of Colorado Health Science Center, Denver, Colo. 106 9L
 cells implanted into the brain of Fischer F344 rats is lethal in 18-28
 days. The MADB106 mammary adenocarcinoma cell line (induced by i.v.
 injection of 9,10-dimethyl-1,2-benzanthracene in a Fischer F344 rat) was
 obtained from Dr. Craig Reynolds of the National Cancer Institute,
 Frederick, M.D. MADB106 cells develop lethal tumors when implanted s.c. or
 i.c. in Fischer 344 rats.
 T9 glioblastoma cells were transduced with a retroviral expression vector
 (LXSN) containing the human cDNA gene for the secreted or
 membrane-associated isoform of M-CSF (obtained from M. R. Jadus). As
 determined by ELISA, clone T9/sM-CSF(H1) secretes M-CSF at a level of 2000
 pg/ml when 1.times.10.sup.6 cells are cultured in 10 ml of media for 3
 days. Secreted M-CSF was shown to be biologically active by its ability to
 induce macrophage colonies from rodent bone marrow samples. Flow
 cytometric analysis indicated that clone T9/mM-CSF(C2) expressed a high
 level of M-CSF on its cell surface, whereas T9/sM-CSF, vector control and
 parental T9 cells did not. Clone T9/mM-CSF(C2) was effectively killed by
 macrophages in an in vitro co-culture experiment but T9/sM-CgF(H1) and
 parental T9 glioblastoma cells were not Clone T9/mM-CSF(C2) and
 T9/sM-CSF(H1) did not differ in their in vitro growth rate, the expression
 of MHC class I or class II antigens, expression of ICAM-1 (CD54), or
 morphology, in comparison with T9 parental or T9/LXSN vector control
 cells.
 Tumor implantation studies were conducted as follows: Animals were
 anesthetized by an intramuscular injection of ketamine (87 mg/kg) and
 xylazine (6.5 mg/kg). A hand-held Dremel drill was used to create a
 shallow depression 3 mm to the right of the sagittal suture and 1 mm
 posterior to the coronal suture. 10 .mu.l tumor cell suspension in PBS was
 injected into the posterior parietal lobe of the brain at a depth of 4 mm
 using a Hamilton syringe, and the needle track was sealed with melted
 parin.
 Animals were implanted intracranially (i.c.) with 1.times.10.sup.5
 T9/mM-CSF or T9/sM-CSF cells. It was found that animals injected with
 T9/sM-CSF have only a slightly longer survival compared to controls
 implanted with T9 parental cells. In contrast, 80% of the animals
 implanted with T9/mM-CSF cells survived. Rejection of mM-CSF cells amongst
 various clones correlated with the level of membrane expression of M-CSF.
 Clone C2 expressed the highest level of M-CSF on its membrane and showed
 73% overall rejection, whereas clone F12 showed a much lower level of
 expression and was rejected in only 20% of animals.
 Animals that survived the i.c. challenges with T9/mM-CSF cells were
 subjected to i.c. rechallenges with 1.times.10.sup.5 parental T9 glioma
 cells or 1.times.10.sup.6 9L glioma cells. The results (illustrated in
 FIG. 6) demonstrate that rejection of T9/mM-CSF cells is followed by a
 long-lasting glioma-associated immunity (.box-solid.) compared with naive
 controls (.circle-solid.). These animals were 100% immune to i.c.
 challenges with parental T9 glioma cells (Panel A) and were partially
 protected from a challenge with 9L glioma cells (Panel B). In contrast,
 the animals were not protected from a challenge with 1.times.10.sup.5
 MADB106 mammary adenocarcinoma cells (Panel C).
 Spleen cell transfer experiments were conducted to determine the origin of
 the acquired tumor-cell resistance. Results (illustrated in FIG. 7, Upper
 Panel) demonstrated that tumor resistance in the mM-CSF treated animals
 could be conferred by cells bearing the pan T-cell marker CD3. Transfer of
 spleen cells from T9/mM-CSF immunized animals into naive recipients
 resulted in immunity to i.c. challenge with 10.sup.5 T9 parental cells
 (.circle-solid.). However, transfer of immune spleen cells depleted of T
 cells by anti-CD3 plus complement did not confer tumor resistance
 (.tangle-solidup.).
 Tumor resistance could also be conferred by administering mM-CSF secreting
 cells at a site distant to the brain (FIG. 7, Lower Panel).
 1.times.10.sup.7 viable T9/mM-CSF cells injected s.c. in the flank were
 rejected in approximately one week. These animals were completely
 resistant to a subsequent i.c. challenge with parental T9 glioma cells
 (.box-solid.). In contrast, animals initially treated with T9/LXSN vector
 control cells (.circle-solid.) were not resistant.
 The combined results indicate that syngeneic mM-CSF expressing tumor cells
 initiate a systemic anti-tumor response that is reactive against tumor
 cells of the same tissue type. The tumor resistance is mediated, at least
 in part, by T lymphocytes.
 Example 6
 Secreted IL-4Expression in a Syngeneic Vaccine Model
 The use of an IL-4 producing cell line in a cancer vaccine was modeled in
 experiments using a Balb/c lymphoma line that secretes IL-4 at high
 concentration.
 In one experiment, mice were coinjected with the lymphoma cells and the
 mouse melanoma cell line B16. Tumor growth was delayed by 2 weeks, in
 comparison with mice injected with B16 cells alone, or B16 cells mixed
 with non-cytokine secreting lymphoma cells. When the B16 cells were
 irradiated before mixing with the IL-4 cells, animals receiving the
 composition had no significant survival advantage against a subsequent
 challenge, compared with controls.
 In another experiment, C57BL/6 mice were coinjected with IL-4 secreting
 lymphoma cells and the carcinoma cell line LL/2. Only one out of three
 mice developed tumor (the others were tumor-free), whereas all three mice
 in group injected with LL/2 alone developed tumor. Histologic examination
 of the one tumor in the treated group demonstrated prominent intratumor
 eosinophilia, as well as prominent splenic eosinophilia. The surviving
 mice are subsequently tested for immunity by rechallengwe with LL/2 cells
 alone.
 Example 7
 Use of Combination Vaccine Comprising Autologous Tumor Cells and IL-4
 Secreting Allogeneic Cells in Human Ovarian Cancer
 This example describes a protocol for the use of a cellular vaccine in the
 treatment of human ovarian cancer. The vaccine comprises various amounts
 of IL-4-secreting 4CI 107 cells (Example 1) mixed with autologous tumor
 cells.
 Women diagnosed with Stage III or Stage IV epithelial ovarian cancer are
 candidates for this protocol. At the primary surgical staging laparotomy
 (day 0), a portion of tumor is collected, reduced to a single cell
 suspension, and used to establish both a primary cell culture and an
 autologous tumor master cell bank. Peripheral blood cells, and lymphocytes
 from the most cephalad para-aortic lymph node, are collected and frozen
 for future in vitro testing. Beginning on the fifth day, patients receive
 four weekly subcutaneous vaccine injections in the anterior thigh Nine
 days after the last injection, patients begin weekly adjuvant
 chemotherapy, consisting of intravenous (i.v.) Cisplatin at 50 mg/m.sup.2
 for nine consecutive weeks.
 A second set of four weekly mixed tumor cell vaccinations begins three
 weeks after the last course of chemotherapy. This set of vaccinations
 consists of the same concentration of cells as the first set. A
 second-look laparotomy is performed three weeks after the last vaccine
 injection. At this time, the surgeon performs an appropriate secondary
 debulking of any gross residual disease, and removes one or two pelvic,
 para-aortic or external iliac lymph nodes. The lymphocytes obtained from
 the draining nodes and the lymphocytes obtained at the end of the
 immunization procedures are compared with lymphocytes obtained at time 0
 for in vitro analysis of the host's immunological response to autologous
 and allogeneic tumor cells as a result of receiving the vaccine. Skin
 testing is also conducted to evaluate the allogeneic and the autologous
 cell-mediated immune reactivity at various intervals during the protocol.
 Subjects:
 Patients are eligible for this study if they fulfill all of the following
 criteria:
 Women at least 18 years of age with histologically confirmed epithelial
 ovarian cancer: F.I.G.O. Stage II and Stage IV. A patient is considered a
 candidate for this protocol even with suboptimally debulked disease.
 Primary staging laparotomy and other procedures are performed according to
 FIGO criteria (DiSaia et al. (1993) Clinical Gynecologic Oncology, St.
 Louis: Mosby--Year Book, pp 682-684).
 Karnofsky Performance Status (DiSaia et al. supra p 375 fl) of at least 60
 percent preoperatively and on the first day of treatment.
 Clinically estimated life expectancy of at least three months.
 Prior to receiving the first vaccination, patients must meet the following
 laboratory criteria: Hematocrit &gt;20; Neutrophil count &gt;1.5.times.10.sup.6/
 ml; Platelet count &gt;100,000; Creatinine &lt;2.0.
 Levels of CA-125 antigen must be &gt;35 U/ml prior to surgery but not prior to
 the first vaccination.
 Patients must provide sufficient tumor mass to generate all eight vaccine
 doses. The autologous tumor cells obtained at the time of debulking must
 be at least 80 percent viable and contain greater than 80 percent tumor
 cells.
 Patients must be willing to participate in the study and provide informed
 consent.
 Patients are ineligible for this study if they fulfill any of the following
 criteria:
 Patients who have received any cytotoxic chemotherapy, radiotherapy or
 cytokine therapy within four weeks of entry.
 Patients with known contraindication to platinum-based cytotoxic
 chemotherapy.
 Patients with autoimmune diseases, lymphoproliferative diseases (including
 other hematologic malformative diseases), serological evidence of HIV
 exposure or any sign of active bacterial or viral infection or fever of
 unknown origin.
 Patients with other concomitant invasive cancer.
 Patients who within two months prior to entry were treated for a duration
 exceeding two weeks with immunosuppressive agents, including but not
 limited to corticosteroids and cyclosporine.
 Patients who have received organ transplants, or who are pregnant or breast
 feeding.
 Patients with psychological or geographic conditions which prevent adequate
 follow-up or compliance with the protocol.
 Patients who are unable to provide informed consent.
 Preparation of the Vaccine:
 The UCI-107 IL-4-E cell line (prepared as described in Example 1) has been
 extensively tested for the presence of microorganisms by our own and
 several independent outside laboratories. The following tests have been
 conducted by the following laboratories, and the results were all
 negative:
 U.C.I. Laboratory: aerobic and anaerobic Bacteria.
 Nichols Laboratory, San Juan Capistrano: mycoplasma pneumonial culture.
 Quality Biotech, Inc., New Jersey: tested cells by DNA PCR for CMV, EBV,
 and HBV.
 Co-cultivation of cells with Mus Dunni cells; amplification of cell
 supernatant with Mus Dunni cells for detection of replication competent
 retroviruses.
 In addition, a mouse antibody production test for viruses is conducted as
 follows: 17/LXSN IL-4 supernatant is injected intraperitoneally into
 mice. 0.7 to 1.0 ml of blood is drawn at four weeks after injection. The
 blood is sent to Dr. Dixie Fisher's laboratory, University of Southern
 California, Vivaria Animal Diagnostic and Research Laboratory, for
 serologic analysis for the following murine virus antibodies: ectromelia
 (integument); GD VII (central nervous system); Lactic dehydrogenase
 elevating virus (liver); Mouse hepatitis virus (digestive tract);
 Pneumonia virus of mice (respiratory tract); Reo-3 (intestinal tract);
 Sendai virus (respiratory tract). Cell supernatant is also tested for
 endotoxin and adventitious viral contaminants (Quality Biotech, Inc.).
 Monolayers of UCI-107 IL-4-E cells for implantation are washed three times
 in PBS and established in Aim V medium. Aim V medium is approved for cell
 culture prior to implantation into patients and contains no bovine serum.
 Cells are passed in Aim V for two weeks before use in the vaccine
 preparations, then washed and resuspended in PBS. Cell viability is
 established by trypan blue exclusion. Cell suspensions with less than 90
 percent viability are not employed.
 Tumors are diagnosed by intraoperative examination of frozen sections. Once
 identified as an epithelial ovarian carcinoma, a portion of viable solid
 tumor tissue is aseptically collected, rapidly transported and processed
 within 4 hours into a single cell suspension by the method of Weisenthal
 et al.
 One aliquot is used in an attempt to establish a primary cell culture. The
 remainder is resuspended in complete medium containing 10% DMSO, and
 aliquoted for storage under liquid nitrogen. The patient identification
 number and date of collection is recorded in a computer database and on
 each vial. The aliquoted frozen tumor cells are used as the autologous
 vaccine component If a cell culture is established, it is used for in
 vitro testing; otherwise primary tumor cells are used. Tumor cells are
 manipulated and stored in AIM V medium plus 15 percent autologous human
 serum.
 Both cells and medium from the autologous tumor master cell bank are tested
 for bacterial contamination at the time the vials are placed into the
 freezer. Samples are inoculated into thioglycolate medium for both aerobic
 and anaerobic bacteria; and streaked on trypticase soy yeast extract
 plates for aerobic bacteria Cultures are incubated for five days at
 37.degree. C. Sample are also tested for endotoxins.
 A 15 ml conical tube containing 10 ml CM and varying doses of UCI-107
 IL-4-E and autologous tumor cells is irradiated for 50 minutes with a
 Cs137 source discharging 200 rads/min. After irradiation, the vaccine
 cells are washed three times in normal saline and injected within 60
 minutes.
 Treatment Plan:
 Three patients are tested for each dose combination, unless the maximum
 tolerated combination, demonstrated by Gade III or greater toxicity or
 irreversible Grade II toxicity is observed. In this case, up to three
 additional patients are entered at the same dose level. If a second
 patient then develops the same degree of toxicity, that dose and cell
 combination is defined as the maximum tolerated combination. If none of
 the three additional patients develop the same degree of toxicity, the
 dose is escalated to the next level until the maximum level to be tested
 is reached. Intrapatient dose escalation is not performed.
 The following table outlines the dosages used:
 TABLE 8
 Number of
 autologous Number of Tumor mass required for
 tumor cells UCI-107 IL-4-E cells Ratio full treatment schedule
 1 .times. 10.sup.7 1 .times. 10.sup.8 1:10 16-20 grams
 5 .times. 10.sup.7 5 .times. 10.sup.7 1:1 80-120 grams
 1 .times. 10.sup.8 1 .times. 10.sup.7 10:1 160-200 grams
 Inoculations are administered subcutaneously 10 cm inferior to the inguinal
 ligament on the anterior mid-thigh. The site of inoculation is alternated
 such that the first inoculation is administered to the left thigh, the
 next to the right thigh, and so on, using a 22 gauge needle.
 The treatment schedule is as follows:
 The date of cytoreductive surgery is marked as day 0.
 Vaccine is given on days 5, 12, 19 and 26.
 Adjuvant Chemotherapy: Nine days after the fourth vaccination, adjuvant
 chemotherapy consisting of i.v. Cisplatin at 50 mg/m.sup.2.times.9 weekly
 cycles is administered (days 35, 42, 49, 56, 63, 70, 77, 84 and 91).
 Cisplatin, combination Cisplatin/Cyclophaphamide, or
 Cisplatin/Cyclophosphamide/ Doxorobicin are believed to be approximately
 equivalent in terms of efficacy and toxicity, even when stratified for
 optimal versus sub-optimal primary debunking. If deemed necessary, in
 addition to the Cisplatin, i.v. Taxol may be used as a salvage therapy.
 Vaccine is given after the chemotherapy on days 112, 119, 126, and 133.
 On day 154, patients undergo a second-look laparotomy that may include
 resection of residual tumor, removal of one or two palpably enlarged
 pelvic or para-aortic lymph nodes or inguinal lymph nodes, if necessary.
 Patients are removed from the study under any of the following conditions:
 Development of any Grade IV toxicity or Grade III toxicity related to
 administration of the vaccine. Grade II skin reactions are expected and
 not a grounds for removal from the study. Grade II reactions include
 localized rash, vaticoma, swelling, or a transient temperature of about
 100.4.degree. C.
 Patient refusal to continue participation.
 Disease Progression--Patients undergo a physical examination and
 determination of CA-125 levels prior to initiation of the chemotherapy and
 every three weeks thereafter. Disease progression is defined as a doubling
 of the CA-125. The post-surgical baseline sample is drawn with the first
 dose of chemotherapy and a doubling must have an absolute level of at
 least 70 U/ml and must be confirmed by repeat sample analysis.
 Immunologic Testing:
 In vitro and in vivo immunological tests are conducted to determine if the
 immunization procedure has induced a host anti-tumor response. Baseline
 reactivity is established before the vaccinations begin. Reactivity
 against the allogeneic cell line serves as a positive control to measure
 patient reactivity against strong antigens.
 The preparation of the specific lymphoid cell populations for immunologic
 testing is performed in the following manner. Peripheral blood mononuclear
 cells (PBMC) are collected from 50 cc of peripheral blood separated by
 FICOLL HYPAQUE.TM. gradients, and stored in liquid nitrogen.
 Lymphocyte cytolytic activity and cytokine production is tested as follows:
 Cytolytic activity of the patient's lymphoid cells is measured in a 4 hour
 .sup.51 Cr release microplate assay and in long-term assays versus the
 allogeneic UCI-107 cell lines, autologous tumor cells, and another ovarian
 tumor cell line from a different patient as a specificity control.
 Delayed Type Hypersensitivity (DTH) is tested by skin reaction. Test
 inoculations include: Mumps (viral), Trichophyton (fungal), PPD
 (bacterial) antigens, 5.times.10.sup.5 irradiated autologous tumor cells,
 or 5.times.10.sup.5 irradiated allogeneic tumor cells. Inoculums are
 administered into the forearm at separately marked sites. The diameter of
 induration is recorded 48 hours following inoculation, and skin test
 responses of less than 5 mm induration are scored as negative. DTH
 responses are assessed prior to vaccination (day 3), prior to chemotherapy
 (day 33), prior to the second series of vaccinations (day 111), and after
 the second series of vaccinations (day 152).
 The ability of the vaccine combinations to improve the outlook of patients
 with ovarian cancer is also assessed according to standard clinical
 criteria
 Although the foregoing invention has been described in some detail by way
 of illustration and example for purposes of clarity and understanding, it
 will be apparent to those skilled in the art that certain changes and
 modifications may be practiced. Therefore, the description and examples
 should not be construed as limiting the scope of the invention, which is
 delineated by the appended claims.