Lymphokine gene therapy of cancer

BACKGROUND
Recent advances in our understanding of the biology of the immune system have lead to the identification of important modulators of immune responses, called cytokines (1-3). Immune system modulators produced by lymphocytes are termed lymphokines, a subset of the cytokines. These agents mediate many of the immune responses involved in anti-tumor immunity. Several of these cytokines have been produced by recombinant DNA methodology and evaluated for their anti-tumor effects. The administration of lymphokines and related immunomodulators has resulted in objective tumor responses in patients with various types of neoplasms (4-7). However, current modes of cytokine administration are frequently associated with toxicities that limit the therapeutic value of these agents.
For example, interleukin-2 (IL-2) is an important lymphokine in the generation of anti-tumor immunity (4). In response to tumor antigens, a subset of lymphocytes termed helper T-cells secrete small quantities of IL-2. This IL-2 acts locally at the site of tumor antigen stimulation to activate cytotoxic T-cells and natural killer cells which mediate systemic tumor cell destruction. Intravenous, intralymphatic and intralesional administration of IL-2 has resulted in clinically significant responses in some cancer patients (4-6). However, severe toxicities (hypotension and adema) limit the dose and efficacy of intravenous and intralymphatic IL-2 administration (5-7). The toxicity of systemically administered lymphokines is not surprising as these agents mediate local cellular interactions and they are normally secreted in only very small quantities.
Additionally, other cytokines, such as interleukin-4 (IL-4), alpha interferon (.alpha.-INF) and gamma interferon (c-INF) have been used to stimulate immune responses to tumor cells. Like IL-2, the current modes of administration have adverse side effects.
To circumvent the toxicity of systemic cytokine administration, several investigators have examined intralesional injection of IL-2. This approach eliminates the toxicity associated with systemic IL-2 administration (8,9,10). However, multiple intralesional injections are required to optimize therapeutic efficacy (9,10). Hence, these injections are impractical for many patients, particularly when tumor sites are not accessible for injection without potential morbidity.
An alternative approach, involving cytokine gene transfer into tumor cells, has resulted in significant anti-tumor immune responses in several animal tumor models (11-14). In these studies, the expression of cytokine gene products following cytokine gene transfer into tumor cells has abrogated the tumorigenicity of the cytokine-secreting tumor cells when implanted into syngeneic hosts. The transfer of genes for IL-2 (11,12) c-INF (13) or interleukin-4 (IL-4) (14) significantly reduced or eliminated the growth of several different histological types of murine tumors. In the studies employing IL-2 gene transfer, the treated animals also developed systemic anti-tumor immunity and were protected against subsequent tumor challenges with the unmodified parental tumor (11,12). Similar inhibition of tumor growth and protective immunity was also demonstrated when immunizations were performed with a mixture of unmodified parental tumor cells and genetically modified tumor cells engineered to express the IL-2 gene. No toxicity associates with localized lymphokine transgene expression was reported in these animal tumor studies (11-14).
While the above gene-transfer procedure has been shown to provide anti-tumor immunity, it still retains practical difficulties. This approach is limited by the inability to transfer functional cytokine genes into many patients' tumor cells, as most patients' tumors cannot be established to grown in vitro and methods for human in vivo gene transfer are not available.
SUMMARY OF THE INVENTION
The present invention demonstrates a novel, more practical method of cytokine cancer immunotherapy. In one approach, selected cells from a patient, such as fibroblasts, obtained, for example, from a routine skin biopsy, are genetically modified to express one or more cytokines. Alternatively, patient cells which may normally serve as antigen presenting cells in the immune system such as macrophages, monocytes, and lymphocytes may also be genetically modified to express one or more cytokines. These modified cells are hereafter called cytokine-expressing cells, ore CE cells. The CE cells are then mixed with the patient's tumor antigens, for example in the form of irradiated tumor cells, or alternatively in the form of purified natural or recombinant tumor antigen, and employed in immunizations, for example subcutaneously, to induce systemic anti-tumor immunity.
The cytokines are locally expressed at levels sufficient to induce or augment systemic anti-tumor immune responses via local immunization at sites other than active tumor sites. Systemic toxicity related to cytokine administration should not occur because the levels of cytokine secreted by the CE cells should not significantly affect systemic cytokine concentrations.
As the amount of cytokine secreted by the CE cells is sufficient to induce anti-tumor immunity but is too low to produce substantial systemic toxicity, this approach provides the benefit of local cytokine administration. In addition, this novel method obviates the need for intralesional injections, which may produce morbidity. Furthermore, the continuous local expression of cytokine(s) at the sites of immunization may also augment anti-tumor immune responses compared to intermittent cytokine injections. This method also provides the advantage of local immunization with the CE cells, as opposed to cumbersome intravenous infusions. This method also eliminates the need for establishing tumor cell lines in vitro as well as transfer of genes into these tumor cells.
This invention also provides an alternative means of localized expression of cytokines to induce and/or increase immune responses to a patient's tumor through genetic modification of cellular expression of both cytokine(s) and tumor antigen(s). In this embodiment, selected cells from a patient are isolated and transduced with cytokine gene(s) as well as gene(s) coding for tumor antigen(s). The transduced cells are called "carrier cells." Carrier cells can include fibroblasts and cells which may normally serve as antigen presenting cells in the immune system such as macrophages, monocytes, and lymphocytes. Transduced carrier cells actively expressing both the cytokine(s) and the tumor antigen(s) are selected and utilized in local immunizations at a site other than active tumor sites to induce anti-tumor immune responses. As with the CE cells, these carrier cells should not produce substantial systemic toxicities, as the levels of cytokine(s) secreted by the carrier cells should not significantly affect systemic cytokine concentrations. This alternate embodiment is advantageous because it obviates the need to obtain samples of the tumor, which is sometimes difficult. However, carrier cells can be utilized in local immunizations in conjunction with tumor cells, tumor cell homogenates, purified tumor antigens, or recombinant tumor antigens to enhance anti-tumor immunity.
Additionally, this second embodiment retains the same advantages as the first embodiment in that the level of cytokine released by the carrier cells is sufficient to induce anti-tumor immunity but is too low to produce substantial systemic toxicity. In addition, as with the first embodiment, this method obviates the need for intralesional injections, and allows for continuous expression of cytokine(s). This method also eliminates the need for establishing continuous cultures in vitro of tumor cells as well as transfer of genes into these tumor cells, and provides the advantage of local immunization with the carrier cells, as opposed to cumbersome lengthy intravenous infusions.
These approaches may also find application in inducing or augmenting immune responses to other antigens of clinical significance in other areas of medical practice.

BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows schematic diagrams of retroviral vectors DC/TKIL2, LXSN-IL2, and LNCX-IL2.
FIG. 2 shows a mean IL-2 concentration of triplicate supernatant samples measured by ELISA. Supernatants were harvested from overnight cultures of approximately 1.5.times.10.sup.6 semi-confluent fibroblasts.
FIG. 3 shows biological activity of the IL-2 secreted by the transduced fibroblasts was demonstrated by measuring mean .sup.3 H-TdR incorporation of an IL-2 dependent T-cell line incubated with triplicate samples of supernatants. Supernatants were harvested from overnight cultures of approximately 1.5.times.10.sup.6 semi-confluent fibroblasts.
FIG. 4 shows comparisons between animals injected with 10.sup.5 CT26 tumor cells alone (.quadrature.); 10.sup.5 CT26 tumor cells and 2.times.10.sup.6 unmodified BALB/C fibroblasts (.box-solid.); 10.sup.5 CT26 tumor cells and 2.times.10.sup.6 IL-2 transduced BALB/C fibroblasts (.oval-solid.); and 10.sup.5 CT26 tumor cells and 1.times.10.sup.6 transduced BALB/C fibroblasts (.largecircle.). Tumor measurements are the mean products of the cross-sectional diameter of the tumors from four animals in each treatment group. The (*) indicates statistically significant difference (P<0.05) in tumor growth curves.
FIG. 5 shows PCR analysis of neomycin phosphotransferase DNA sequences. Lane 1--positive control pLXSN-RI-IL2. Lanes 2 through 4 tests genomic DNA; Lanes 5 and 6 ovary genomic DNA; Lane 7 negative control, no DNA. Identical results were obtained with liver, spleen and lung genomic DNA (data not shown).
FIG. 6 shows the effect of IL-2 modified fibroblasts on tumor establishment and development using 2.times.10.sup.6 fibroblasts mixed with 5.times.10.sup.4 CT26 tumor cells concentrating on the rate of tumor growth.
FIG. 7 shows the effect of IL-2 modified fibroblasts on tumor establishment and development using 2.times.10.sup.6 fibroblasts mixed with 5.times.10.sup.4 CT26 tumor cells concentrating on the time of tumor onset for the individual animal in each treatment group.
FIG. 8 shows the effect of IL-2 modified fibroblasts on tumor establishment and development using 2.times.10.sup.6 fibroblasts mixed with 1.times.10.sup.5 CT26 tumor cells concentrating on the rate of tumor growth.
FIG. 9 shows the effect of IL-2 modified fibroblasts on tumor establishment and development using 2.times.10.sup.6 fibroblasts mixed with 1.times.10.sup.5 CT26 tumor cells concentrating on the time of tumor onset for the individual animal in each treatment group.
FIG. 10 shows the effect of IL-2 modified cells on tumor establishment and development using 2.times.10.sup.6 DCTK-IL2-modified CT26 tumor cells mixed with 1.times.10.sup.5 unmodified CT26 compared to 2.times.10.sup.6 DCTK-IL2-modified fibroblasts mixed with 1.times.10.sup.5 CT26 concentrating on the rate of tumor growth.
FIG. 11 shows the effect of IL-2 modified cells on tumor establishment and development using 2.times.10.sup.6 DCTK-IL2-modified CT26 tumor cells mixed with 1.times.10.sup.5 unmodified CT26 compared to 2.times.10.sup.6 DCTK-IL2-modified fibroblasts mixed with 1.times.10.sup.5 CT26 concentrating on the time of tumor onset for the individual animal in each treatment group.
FIG. 12 shows the effect of IL-2 modified fibroblasts on induction of systemic anti-tumor immunity and the rate of tumor growth. Mice were immunized with 2.times.10.sup.6 fibroblasts mixed with 2.5.times.10.sup.5 irradiated CT26 tumor cells 7 days prior to challenge with 5.times.10.sup.4 fresh tumor cells.
FIG. 13 shows the effect of IL-2 modified fibroblasts on induction of systemic anti-tumor immunity and the time of tumor onset for the individual animal in each treatment group. Mice were immunized with 2.times.10.sup.6 fibroblasts mixed with 2.5.times.10.sup.5 irradiated CT26 tumor cells 7 days prior to challenge with 5.times.10.sup.4 fresh tumor cells.
FIG. 14 shows the effect of IL-2 modified fibroblasts on induction of systemic anti-tumor immunity and the rate of tumor growth. Mice were immunized with 2.times.10.sup.6 fibroblasts mixed with 2.5.times.10.sup.5 irradiated CT26 tumor cells 14 days prior to challenge with 5.times.10.sup.4 fresh tumor cells.
FIG. 15 shows the effect of IL-2 modified fibroblasts on induction of systemic anti-tumor immunity and the time of tumor onset for the individual animal in each treatment group. Mice were immunized with 2.times.10.sup.6 fibroblasts mixed with 2.5.times.10.sup.5 irradiated CT26 tumor cells 14 days prior to challenge with 5.times.10.sup.4 fresh tumor cells.

DETAILED DESCRIPTION
A novel method of tumor immunotherapy is described comprising the genetic modification of cells resulting in the secretion of cytokine gene products to stimulate a patient's immune response to tumor antigens. "Gene" is defined herein to be a nucleotide sequence encoding the desired protein. In one embodiment, autologous fibroblasts genetically modified to secrete at least one cytokine gene product are utilized to immunize the patient in a formulation with tumor antigens at a site other than an active tumor site. In another embodiment, cells genetically modified to express at least one tumor antigen gene product and to secrete at least one cytokine gene product are utilized in formulation to immunize the patient at a site other than an active tumor site. Cytokines are preferably expressed in cells which efficiently secrete these proteins into the surrounding milieu. Fibroblasts are an example of such cells. Fibroblasts or other cells can be genetically modified to express and secrete one or more cytokines, as described later in this specification.
Tumor antigens can be provided by several methods, including, but not limited to the following: 1) CE cells can be transduced with gene(s) coding for tumor antigens. These "carrier cells" are then utilized in patient immunizations. 2) Cloned gene sequences coding for appropriate tumor antigens can be transferred into cells such as fibroblasts or antigen-presenting cells. These cells are then mixed with CE or carrier cells to immunize the patient. 3) Tumor antigens can be cloned in bacteria or other types of cells by recombinant procudures. These antigens are then purified and employed an immunization with CE and/or carrier cells. 4) Tumor antigens can be purified from tumor cells and used, along with CE or carrier cells, to immunize the patient. 5) Tumor cells may be irradiated or mechanically disrupted and mixed with CE and/or carrier cells for patient immunizations.
This invention encompasses the following steps: (A) isolation of appropriate cells for generation of CE cells or carrier cells; (B) isolation of cytokine genes or isolation of cytokine genes and tumor antigen genes, as well as appropriate marker and/or suicide genes; (C) transfer of the genes from (B) to produce the CE cells or carrier cells; (D) preparation of immunological samples of the patient's tumor antigens or other suitable tumor antigens for immunization with CE or carrier cells; (E) inactivation of the malignant potential of tumor cells if they are used as a source of tumor antigens for immunization; and (F) preparation of samples for immunization. Following are several embodiments contemplated by the inventors. However, it is understood that any means known by those in the art to accomplish these steps will be usable in this invention.
(A) Isolation of Cells to Generate CE and Carrier Cells
Cells to be utilized as CE cells and carrier cells can be selected from a variety of locations in the patient's body. For example, skin punch biopsies provide a readily available source of fibroblasts for use in generating CE cells, with a minimal amount of intrusion to the patient. Alternatively, these fibroblasts can be obtained from the tumor sample itself. Cells of hematopoietic origin may be obtained by venipuncture, bone marrow aspiration, lymph node biopsies, or from tumor samples. Other appropriate cells for the generation of CE or carrier cells can be isolated by means known in the art. Non-autologous cells similarly selected and processed can also be used.
(B) Isolation of Genes
Numerous cytokine genes have been cloned and are available for use in this protocol. The genes for IL-2, c-INF and other cytokines are readily available (1-5, 11-14). Cloned genes of the appropriate tumor antigens are isolated according to means known in the art.
Selectable marker genes such as neomycin resistance (Neo.sup.R) are readily available. Incorporation of a selectable marker gene(s) allows for the selection of cells that have successfully received and express the desired genes. Other selectable markers known to those in the art of gene transfer may also be utilized to generate CE cells or carrier cells expressing the desired transgenes.
"Suicide" genes can be incorporated into the CE cells or carrier cells to allow for selective inducible killing after stimulation of the immune response. A gene such as the herpes simplex virus thymidine kinase gene (TK) can be used to create an inducible destruction of the CE cells or carrier cells. When the CE cells or carrier cells are no longer useful, a drug such as acyclovir or gancyclovir can be administered. Either of these drugs will selectively kill cells expressing TK, thus eliminating the implanted transduced cells. Additionally, a suicide gene may be a gene coding for a non-secreted cytotoxic polypeptide attached to an inducible promoter. When destruction of the CE or carrier cells is desired, the appropriate inducer of the promoter is administered so that the suicide gene is induced to produce cytotoxic polypeptide which subsequently kills the CE or carrier cell. However, destruction of the CE or carrier cells may not be required.
Genes coding for tumor antigen(s) of interest can be cloned by recombinant methods. The coding sequence of an antigen expressed by multiple tumors may be utilized for many individual patients.
(C) Transfer of Genes
Numerous methods are available for transferring genes into cultured cells (15). For example, the appropriate genes can be inserted into vectors such as plasmids or retroviruses and transferred into the cells. Electroporation, lipofection and a variety of other methods are known in the field and can be implemented.
One method for gene transfer is a method similar to that employed in previous human gene transfer studies, where tumor infiltrating lymphocytes (TILs) were modified by retroviral gene transduction and administered to cancer patients (16). In this Phase I safety study of retroviral mediated gene transfer, TILs were genetically modified to express the Neomycin resistance (Neo.sup.R) gene. Following intravenous infusion, polymerase chain reaction analyses consistently found genetically modified cells in the circulation for as long as two months after administration. No infectious retroviruses were identified in these patients and no side effects due to gene transfer were noted in any patients (16). These retroviral vectors have been altered to prevent viral replication by the deletion of viral gag, pol and env genes.
When retroviruses are used for gene transfer, replication competent retroviruses may theoretically develop by recombination between the retroviral vector and viral gene sequences in the packaging cell line utilized to produce the retroviral vector. We will use packaging cell lines in which the production of replication competent virus by recombination has been reduced or eliminated. Hence, all retroviral vector supernatants used to infect patient cells will be screened for replication competent virus by standard assays such as PCR and reverse transcriptase assays (16). Furthermore, exposure to replication competent virus may not be harmful. In studies of subhuman primates injected with a large inoculum of replication competent murine retrovirus, the retrovirus was cleared by the primate immune system (17). No clinical illnesses or sequelae resulting from replication competent virus have been observed three years after exposure. In summary, it is not expected that patients will be exposed to replication competent murine retrovirus and it appears that such exposure may not be deleterious (17).
(D) Preparation of Immunological Samples of the Patient's Tumor Antigens or Purified Recombinant Tumor Antigens
Tumor cells bearing tumor associated antigens are isolated from the patient. These cells can derive either from solid tumors or from leukemic tumors. For solid tumors, single-cell suspensions can be made by mechanical separation and washing of biopsy tissue (18).
Hematopoietic tumors may be isolated from peripheral blood or bone marrow by standard methods (19).
A second variant is the use of homogenates of tumor cells. Such homogenates would contain tumor antigens available for recognition by the patient's immune system upon stimulation by this invention. Either unfractionated cell homogenates, made, for example, by mechanical disruption or by freezing and thawing the cells, or fractions of homogenates preferably with concentrated levels of tumor antigens, can be used.
Likewise, purified tumor antigens, obtained for example by immunoprecipitation or recombinant DNA methods, could be used. Purified antigens would then be utilized for immunizations together with the CE cells and/or carrier cells described above to induce or enhance the patient's immune response to these antigens.
In the embodiments employing carrier cells, tumor antigens are available through their expression by the carrier cells. These carrier cells can be injected alone or in conjunction with other tumor antigen preparations or CE cells. Likewise, when CE cells are used, purified recombinant tumor antigen, produced by methods known in the art, can be used.
If autologous tumor cells are not readily available, heterologous tumor cells, their homogenates, their purified antigens, or carrier cells expressing such antigens could be used.
(E) Inactivation of Tumor Cells
When viable tumor cells are utilized in immunizations as a source of tumor antigens, the tumor cells can be inactivated so that they do not grow in the patient. Inactivation can be accomplished by several methods. The cells can be irradiated prior to immunization (18). This irradiation will be at a level which will prevent their replication. Such viable calls can then present their tumor antigens to the patient's immune system, but cannot multiply to create new tumors.
Alternatively, tumor cells that can be cultured may be transduced with a suicide gene. As described above, a gene such as the herpes simplex thymidine kinase (TK) gene can be transferred into tumor cells to induce their destruction by administration of acyclovir or gancyclovir. After immunization, the TK expressing tumor cells can present their tumor antigens, and are capable of proliferation. After a period of time during which the patients's immune response is stimulated, the cells can be selectively killed. This approach might allow longer viability of the tumor cells utilized for immunizations, which may be advantageous in the induction or augmentation of anti-tumor immunity.
(F) Preparation of Samples for Immunization
CE cells and/or carrier cells and tumor cells, and/or homogenates of tumor cells and/or purified tumor antigen(s), are combined for patient immunization. Approximately 10.sup.7 tumor cells will be required. If homogenates of tumor cells or purified or non-purified fractions of tumor antigens are used, the tumor dose can be adjusted based on the normal number of tumor antigens usually present on 10.sup.7 intact tumor cells. The tumor preparation should be mixed with numbers of CE or carrier cells sufficient to secrete cytokine levels that induce anti-tumor immunity (11-12) without producing substantial systemic toxicity which would interfere with therapy.
The cytokines should be produced by the CE cells or the carrier cells at levels sufficient to induce or augment immune response but low enough to avoid substantial systemic toxicity. This prevents side effects created by previous methods' administration of greater than physiological levels of the cytokines.
These mixtures, as well as carrier cells that are utilized alone, will be formulated for injection in any manner known in the art acceptable for immunization. Because it is important that at least the CE cells and carrier cells remain viable, the formulations must be compatible with cell survival. Formulations can be injected subcutaneously, intramuscularly, or in any manner acceptable for immunization.
Contaminants in the preparation which may focus the immune response on undesired antigens should be removed prior to the immunizations.
The following examples are provided for illustration of several embodiments of the invention and should not be interpreted as limiting the scope of the invention.
EXAMPLE I
Immunization with Fibroblasts Expressing IL-2 Mixed with Irradiated Tumor Cells
1) Isolation of Autologous Fibroblasts for Use in Generating IL-2 Secreting CE Cells
Skin punch biopsies will be obtained from each patient under sterile conditions. The biopsy tissue will be minced and placed in RPMI 1640 media containing 10% fetal calf serum (or similar media) to establish growth of the skin fibroblasts in culture. The cultured fibroblasts will be utilized to generate IL-2 secreting CE cells by retroviral mediated IL-2 gene transfer.
2) Retrovital Vector Preparation and Generation of IL-2 Secreting CE Cells
The cultured skin fibroblasts will then be infected with a retroviral vector containing the IL-2 and Neomycin resistance (Neo.sup.R) genes. An N2 vector containing the Neo.sup.R gene will be used, and has been previously utilized by a number of investigators for in vitro and in vivo work, including investigations with human subjects (16). The IL-2 vector will be generated from an N2-derived vector, LLRNL, developed and described by Friedmann and his colleagues (20). It will be made by replacement of the luciferase gene of LLRNL with a full-length cDNA encoding human IL-2. Retroviral vector free of contaminating replication-competent virus is produced by transfection of vector plasmid constructions into the helper-free packaging cell line PA317. Before infection of patients' cells, the vector will have been shown to be free of helper virus. In the event that helper virus is detected, the vector will be produced in the GP+envAM12 packaging cell line in which the viral gag and pol genes are separated from the env, further reducing the likelihood of helper virus production.
3) Transduction Protocol
The cultured primary fibroblasts will be incubated with supernatant from the packaging cell line as described (20). Supernatant from these cells will be tested for adventitious agents and replication competent virus as described (16) and outlined in Table 1. The fibroblasts are washed and then grown in culture media containing G418, (a neomycin analogue) to select for transduced cells expressing the Neo.sup.R gene. The G418-resistant cells will be tested for expression of the IL-2 gene by measuring the concentration of IL-2 in the culture supernatant by an enzyme linked immunosorbent assay (ELISA) (12). G418-resilient cells expressing IL-2 will be stored at -70.degree. C. until required for subsequent use in immunizations.
TABLE 1______________________________________Adventitious Agents and Safety Testing______________________________________ 1. Sterility 2. Mycoplasma 3. General Safety 4. Viral Testing LCM Virus Thymic agent S+/L- eco S+/L- xeno S+/L- ampho 3T3 amplification MRC-5/Vero______________________________________
4) Preparation of Irradiated Tumor Cells
Tumors obtained form clinically indicated surgical resections or from superficial lymph node or skin metastases will be minced into 2-3 mm pieces and treated with collagenase and DNAse to facilitate separation of the tumor into a single cell suspension. The collected cells will be centrifuged and washed in RPMI 1640 media and then cryopreserved in a solution containing 10% dimethyl sulphoxide and 50% fetal calf serum in RPMI 1640 media. The cells will be stored in liquid nitrogen until the time of administration. Prior to their use in subcutaneous immunizations, the cells will be thawed, washed in media free of immunogenic contaminants, and irradiated with 4,000 rads per minute for a total of 20,000 rads in a cesium irradiator.
5) Patient Selection
Patients will have a histologically confirmed diagnosis of cancer. Patients with tumors that must be resected for therapeutic purposes or with tumors readily accessible for biopsy are most appropriate for this embodiment of the invention.
6) Pretreatment Evaluation
The following pretreatment evaluations will be performed:
1) History and physical examination including a description and quantification of disease activity.
2) Performance Status Assessment
0=Normal, no symptoms
1=Restricted, but ambulatory
2=Up greater than 50% of waking hours, capable of self-care
3=Greater than 50% of waking hours confined to bed or chair, limited self-care
4=Bedridden
3) Pretreatment Laboratory:
CBC with differential, platelet count, PT, PTT, glucose, BUN, creatinine, electrolytes, SGOT, SGPT, LDH, alkaline phosphatase, bilirubin, uric acid, calcium, total protein albumin.
4) Other Analyses:
Urinalysis
CH.sub.50, C.sub.3 and C.sub.4 serum complement levels Immunophenotyping of peripheral blood B cell and T cell subsets
Assays for detectable replication-competent virus in peripheral blood cells
PCR assays of peripheral blood leukocytes for Neo.sup.R, IL-2 and viral env
5) Other Pretreatment Evaluation:
Chest X-ray and other diagnostic studies including computerized tomography (CT), magnetic resonance imaging (MRI) or radionuclide scans may be performed to document and quantify the extent of disease activity.
Follow-up evaluations of these assessments at regular intervals during the course of therapy (approximately every 1 to 3 months) will be useful in determining response to therapy and potential toxicity, permitting adjustments in the number of immunizations administered.
7) Restrictions on Concurrent Therapy
For optimal effects of this treatment, patients should receive no concurrent therapy which is known to suppress the immune system.
8) Final Formulation
Each patient will receive subcutaneous immunizations with a mixture if irradiated tumor cells and autologous fibroblast CE cells genetically modified to secrete IL-2. Approximately 10.sup.7 tumor cells will be mixed with 10.sup.7 fibroblasts known to secrete at least 20 units/ml of IL-2 in tissue culture when semi-confluent (12). The irradiated tumor cells and genetically modified fibroblasts will be placed in a final volume of 0.2 ml normal saline for immunization.
9) Dose Adjustments
At least two subcutaneous immunizations will be administered, two weeks apart, with irradiated tumor cells and autologous fibroblasts genetically modified to secrete IL-2. If no toxicity is observed, subsequent booster immunizations may be administered periodically (at least one week apart) to optimize the anti-tumor immune response.
J) Treatment of Potential Toxicity
Toxic side effects are not expected to result from these immunizations. However, potential side effects of these immunizations are treatable in the following manner:
If massive tumor cell lysis results, any resulting uric acid nephropathy, adult respiratory distress syndrome, disseminated intravascular coagulation or hyperkalemia will be treated using standard methods.
Local toxicity at the sites of immunization will be treated with either topical steroids and/or surgical excision of the injection site as deemed appropriate.
Hypersensitivity reactions such as chills, fever and/or rash will be treated symptomatically with antipyretics and antihistamines. Patients should not be treated prophylactically. Should arthralgias, lymphadenopathy or renal dysfunction occur, treatment with corticosteroids and/or antihistamines will be instituted. Anaphylaxis will be treated by standard means such as administration of epinephrine, fluids, and steroids.
EXAMPLE II
A. Retroviral IL-2 Gene Transfer and Expression in Fibroblasts
Retroviral vectors were employed to transfer and express IL-2 and neomycin phosphotransferase genes in murine and primary human fibroblasts. The retroviral vector DC/TKIL2 produced by Gilboa and co-workers (Gansbacher, et al., J. Exp. Med. 172:1217-1223, 1990, which is incorporated herein by reference) was utilized to transduce murine fibroblasts for application in an animal tumor model (see Section B below). Human fibroblasts were transduced with the retroviral vector LXSN-RI-IL2 (SEQ ID NO:1). Schematic diagrams of the structure of these retroviral vectors are provided in FIG. 1. A more complete description of the LXSN-RI-IL2 vector, including its nucleotide sequence, is provided in Example III and in Tables 2, 3, 4, 5 and 6 (SEQ ID NO:1).
Following infection with the described vectors and selection for 2-3 weeks in growth media containing the neomycin analogue G418, Balb/c and human embryonic fibroblast culture supernatants were harvested and tested for IL-2 by an enzyme-linked immunosorbent assay (ELISA). FIG. 2 depicts the levels of IL-2 secreted by the transduced fibroblasts.
These results can be confirmed using negative control fibroblasts infected with an N2-derived retroviral vector expressing an irrelevant gene such as luciferase or .beta.-galactosidase and studies with adult human fibroblasts.
Biological activity of the IL-2 expressed by the transduced human fibroblasts was confirmed by a cell proliferation bioassay employing an IL-2 dependent T cell line. In this assay, supernatant from the transduced fibroblasts and control unmodified fibroblasts were incubated with the IL-2 dependent T cell line CTLL-2. Incorporation of .sup.3 H-thymidine was measured as an indicator of cell proliferation and IL-2 activity (FIG. 3).
B. Efficacy of Transduced Fibroblasts in an Animal Tumor Model
The efficacy of fibroblasts genetically modified to secrete IL-2 was tested in an animal model of colorectal carcinoma. In these studies, the Balb/c CT26 tumor cell line was injected subcutaneously with Balb/c fibroblasts transduced to express IL-2. Control groups included animals injected with 1) a mixture of CT26 tumor cells and unmodified fibroblasts; 2) CT26 tumor cells without fibroblasts and 3) transduced fibroblasts alone. No tumors were detected in 3/8 animals treated with transduced fibroblasts and CT26 cells. In contrast, all untreated control animals (8/8) injected with CT26 tumor cells developed palpable tumors. No tumors were detected in the animals inoculated with transduced fibroblasts without CT26 tumor cells. The mean CT26 tumor size in Balb/c mice injected with the IL-2 secreting fibroblasts was considerably smaller compared to the control groups (FIG. 4). A multivariate non-parametric statistical procedure (Koziol, et al., Biometries 37:383-390, 1981 and Koziol, et al., Computer Prog. Biomed. 19:69-74, 1984, which is incorporated herein by reference) was utilized to evaluate differences in tumor growth among the treatment groups. The tumor growth curves for the four treatment groups presented in FIG. 4 were significantly different (p=0.048). Subsequent comparisons between treatment groups revealed a significant difference (p<0.05) in tumor growth between animals injected with CT26 tumor cells alone and animals treated with 2.times.10.sup.6 transduced fibroblasts and CT26 tumor cells (FIG. 4).
EXAMPLE III
A. Project Overview
Lymphokine gene therapy of cancer will be evaluated in cancer patients who have failed conventional therapy. An N2-derived vector containing the neomycin phosphotransferase gene will be used. This vector has been employed by a number of investigators for in vitro and in vivo studies including recently approved investigations with human subjects (Rosenberg et al., N. Eng. J. Med., 323:570-578, 1990). The lymphokine vectors used in this investigation will be generated from the N2-derived vector, LXSN, developed and described by Miller et al., Mol. Cell Biol. 6:2895, 1986 and Miller et al., BioTechniques 7:980, 1989, which are incorporated herein by reference. The vector LXSN-RI-IL2 contains human IL-2 cDNA under the control of the retroviral 5' LTR promoter and the neomycin phosphotransferase gene under the control of the SV40 promoter (see FIG. 1). The normal human IL-2 leader sequence has been replaced with a chimeric sequence containing rat insulin and human IL-2 leader sequences (see Tables 2, 3, 4, 5 and 6 (SEQ ID NO:1)). This chimeric leader sequence enhances IL-2 gene expression.
To construct the LXSN-RI-IL2 vector, the bacterial plasmid pBC12/CMV/IL2 (Cullen, B. R., DNA 7:645-650, 1988, which is incorporated herein by reference) containing the full-length IL-2 cDNA and chimeric leader sequence was digested with HindIII and the ends were blunted using Klenow polymerase. IL-2 cDNA was subsequently released from the plasmid by digestion with BamHI. The IL-2 fragment was purified by electrophoresis in a 1% agarose gel and the appropriate band was extracted utilizing a glass powder method. Briefly, the gel slice was dissolved in 4M NaI at 55.degree.. After cooling to room temperature, 4 .mu.l of oxidized silica solution (BIO-101, La Jolla, Calif.) was added to adsorb the DNA. The silica was then washed with a cold solution of 50% ethanol containing 0.1M NaCl in TE buffer. The DNA was eluted from the silica by heating at 55.degree. in distilled H.sub.2 O. The purified IL-2 cDNA was then directionally ligated into the HpaI-BamHI cloning sites of the pLXSN vector. A more complete description of the pLXSN-RI-IL2 vector and its partial nucleotide sequence are provided in Tables 2, 3, 4, 5 and 6 (SEQ ID NO:1).
TABLE 2______________________________________Description of the LXSN-RI-IL2from position 1 to 6365Bases Description______________________________________ 1-589 Moloney murine sarcoma virus 5' LTR 659-1458 The sequence of the extended packaging signal1469-2151 IL-2 cDNA with chimeric leader sequencc1469-1718 IL-2 chimeric leader sequence1647-1718 coding region of the signal peptide1719-2151 Mature IL-2 coding sequence2158-2159 Mo mu sarcoma virus end/SV 40 start2159-2503 Simian virus 40 early promoter2521-2522 Simian virus DNA end/Tn5 DNA start2557-3351 Neomycin phosphotransferase3370-3371 Tn5 DNA end/Moloney murine leukemia virus start3411-4004 Moloney murine leukemia virus 3' LTR4073-4074 Moloney murine leukemia DNA end/pBR322 DNA start4074-6365 Plasmid backbone______________________________________
TABLE 3__________________________________________________________________________Enzyme (# Cuts) Position(s)__________________________________________________________________________Aat1 (2) 1961, 2481Aat2 (2) 811, 6295Acc1 (1) 4252Acc2 (19) 392, 394, 445, 969, 971, 1193, 2751, 3052, 3084, 3807, 3809, 4081, 4083. 4186, 4527, 5108, 5438, 5931, 6263Acy1 (5) 808, 2685, 3860, 5910, 6292Afl1 (13) 260, 273, 328, 626, 756, 1277, 3201, 3676, 3689, 3744, 4041, 5511, 5733Afl2 (4) 34, 1064, 1955, 3446Afl3 (2) 1592, 4480Aha1 (20) 161, 237, 473, 474, 602, 644, 789, 2689, 2849, 3578, 3653, 3888, 3889, 4017, 4059, 4126, 4161, 4860, 5556, 5907Aha2 (5) 808, 2685, 3860, 5910, 6292Aha3 (3) 5239, 5258, 5950Alu1 (33) 29, 33, 119, 190, 411, 654, 734, 742, 1470, 1486, 1751, 1935, 2003, 2446, 2500, 2791, 3249, 3441, 3445, 3532, 3607, 3826, 4069, 4122, 4141, 4422, 4648, 4738, 4784, 5041, 5562, 5662, 5725Alw1 (20) 1110, 1414, 1665, 2018, 2147, 2160, 2529, 2553, 2864, 2929, 3110, 4027, 5041, 5127, 5129, 5225, 5226, 5689, 6006, 6010AlwN1 (4) 231, 3572, 3647, 4896Aoc1 (2) 847, 1076Aoc2 (19) 323, 413, 426, 597, 1583, 1721, 2631, 2724, 2798, 2988, 3050, 3739, 3828, 3841, 4012, 4300, 4798, 5959, 6044Aos1 (2) 2787, 5595ApaL1 (4) 1717, 4296, 4794, 6040Apy1 (22) 315, 623, 801, 814, 1227, 1252, 1275, 1295, 1325, 1526, 1536, 1558, 1630, 2196, 2251, 2268, 3072, 3731, 4038, 4508, 4629, 4642Aqu1 (6) 241, 472, 1998, 3821, 3854, 3887Ase1 (2) 1801, 5545Asp700 (1) 5972Asp7l8 (2) 476, 3891AspA1 (1) 1145Asu1 (29) 169, 200, 245, 260, 273, 328, 626, 756, 826, 839, 1043, 1254, 1277, 1532, 1649, 3201, 3541, 3586, 3616, 3661, 3676, 3689, 3744, 4041, 5415, 5494, 5511, 5733, 6349Ava1 (6) 241, 472, 1998, 3821, 3854, 3887Ava2 (13) 260, 273, 328, 626, 756, 1277, 3201, 3676, 3689, 3744, 4041, 5511, 5733Ava3 (2) 2232, 2304Avr2 (2) 1962, 2482Bal1 (3) 658, 1169, 2767BamH1 (1) 2152Ban1 (9) 318, 476, 1200. 2684, 2719, 3734, 3859, 3891, 5321Ban2 (8) 413, 426, 597, 1583, 3050, 3828, 3841, 4012Bbe1 (2) 2688, 3863Bbv1 (22) 969, 997, 1738, 2493, 2632, 2758, 2800, 2816, 2909, 3321, 4060, 4131, 4228, 4372, 4390, 4809, 4899, 4902, 5108, 5411, 5600, 5802Bcl1 (1) 2526Bgl1 (2) 2435, 5493Bsp1286I (19) 323, 413, 426, 597, 1583, 1721, 2631, 2724, 2798, 2988, 3050, 3739, 3828, 3841, 4012, 4300, 4798, 5959, 6044BspH1 (3) 5200, 6208, 6313BspM1 (4) 150l, 2500, 2572, 2953BssH2 (4) 392, 443, 3082, 3807BstE2 (1) 1145BstN1 (22) 315, 623, 801, 814, 1227, 1252, 1275, 1295, 1325, 1526, 1536, 1558, 1630, 2196, 225l, 2268, 3072, 3731, 4038, 4508, 4629, 4642BstU1 (19) 392, 394, 445, 969, 971, 1193, 2751, 3052, 3084, 3807, 3809, 4081, 4083, 4186, 4527, 5108, 5438, 5931, 6263BstX1 (1) 2060BstY1 (ll) 2010, 2152, 2521, 2856, 3102, 5121, 5132, 5218, 5230, 5998, 6015Bsu36I (2) 847, 1076Ccr1 (1) 1998Cfo1 (31) 394, 396, 445, 447, 714, 971, 2679, 2687, 2751, 2788, 3054, 3084, 3086, 3314, 3809, 3811, 3862, 4083, 4186, 4216, 4357, 4390, 4660, 4727, 4827, 5001, 5110, 5503, 5596, 5933, 6265Cfr1 (9) 656, 790, 1167, 1188, 2591, 2765, 3156, 3183, 5761Cfr10I (3) 3004, 3185, 5453Cfr13X (29) 169, 200, 245, 260, 273, 328, 626, 756, 826, 839, 1043, 1254, 1277, 1532, 1649, 3201, 3541, 3586, 3616, 3661, 3676, 3689, 3744, 4041, 5415, 5494, 5511, 5733, 6349Cvn1 (2) 847, 1076Dde1 (23) 75, 165, 191, 282, 553, 847, 1076, 1348, 1692, 2442, 3348, 3487, 3582, 3657, 3698, 3879, 3967, 4290, 4755, 5164, 5330, 5870, 6296Dpn1 (30) 95, 1104, 1236, 1421, 1659, 2012, 2154, 2523, 2528, 2547, 2858, 2936, 3017, 3026, 3104, 3507, 4021, 5048, 5123, 5134, 5142, 5220, 5232, 5337, 5678, 5696, 5742, 6000, 6017, 6053Dra1 (3) 5239, 5258, 5950Dra2 (4) 328, 1277, 3744, 6349Eae1 (9) 656, 790, 1167, 1108, 2591, 2765, 3156, 3183, 5761Eag1 (2) 790, 2591Eco47I (13) 260, 273, 328, 626, 756, 1277, 3201, 3676, 3689, 3744, 4041, 5511, 5733Eco52I (2) 790, 259lEco81X (2) 847, 1076EcoN1 (2) 850, 1450Eco0109I (4) 328, 1277, 3744, 6349EcoR1 (1) 1460EcoR1* (14) 938, 1037, 1460, 1798, 1805, 1928, 2064, 2121, 2236, 2308, 2400, 5240, 5546, 5801EcoR2 (22) 313, 621, 799, 812, 1225, 1250, 1273, 1293, 1323, 1524, 1534, 1556, 1628, 2194, 2249, 2266, 3070, 3729, 4036, 4506, 4627, 4640EcoR5 (4) 137, 213, 3554, 3629EcoT22I (2) 2232, 2304Fdi2 (2) 2787, 5595Fnu4H1 (41) 793, 967, 983, 986, 1191, 1752, 2430, 2507, 2594, 2646, 2657, 2747, 2752, 2789, 2830, 2917, 2920, 2923, 3159, 3255, 3296, 3310, 4074, 4120, 4217, 4270, 4386, 4404, 4407, 4525, 4680, 4823, 4888, 4891, 5097, 5425, 5614, 5764, 5791, 5886, 6115FnuD2 (9) 392, 394, 445, 969, 971, 1193, 2751, 3052, 3084, 3807, 3809, 4081, 4083, 4186, 4527, 5108, 5438, 5931, 6263Fok1 (13) 498, 1198, 1358, 1679, 2333, 2552, 3009, 3034, 3912, 4168, 5339, 5520, 5807Fsp1 (2) 2787, 5595Hae2 (4) 2688, 3863, 4358, 4728Hae3 (35) 171, 202, 247, 658, 792, 828, 840, 1045, 1169, 1190, 1255, 1534, 1650, 1866, 1961, 2423, 2429, 2438, 2481, 2593, 2767, 3158, 3185, 3543, 3588, 3618, 3663, 4495, 4506, 4524, 4958, 5416, 5496, 5763, 6350Hap2 (30) 161, 237, 473, 601, 643, 789, 2590, 2667, 2689, 2717, 2848, 2938, 3005, 3186, 3578, 3653, 3888, 4016, 4058, 4126, 4160, 4687, 4834, 4860, 5050, 5454, 5488, 5555, 5665, 5907Hga1 (8) 455, 707, 960, 1580, 4175, 4591, 5169, 5899HgiA1 (9) 413, 1721, 2798, 2988, 3828, 4300, 4798, 5959, 6044Hha1 (31) 394, 396, 445, 447, 714, 971, 2679, 2687, 2751 2788, 3054, 3084, 3086, 3314, 3809, 3811, 3862, 4083, 4186, 4216, 4357, 4390, 4660, 4727, 4827, 5001, 5110, 5503, 5596, 5933, 6265HinP1 (31) 392, 394, 443, 445, 712, 969, 2677, 2685, 2749, 2786, 3052, 3082, 3084, 3312, 3807, 3809, 3860, 4081, 4184, 4214, 4355, 4388, 4658, 4725, 4825, 4999, 5108, 5501, 5594, 5931, 6263Hinc2 (1) 5914Hind2 (1) 5914Hind3 (1) 2498Hinf1 (14) 298, 517, 857, 868, 1553, 1814, 3170, 3304, 3356, 3881, 4380, 4455, 4851, 5368Hpa2 (30) 161, 237, 473, 601, 643, 789, 2590, 2667, 2689, 2717, 2848, 2938, 3005, 3186, 3578, 3653, 3888, 4016, 4058, 4126, 4160, 4687, 4834, 4860, 5050, 5454, 5488, 5555, 5665, 5907Hph1 (11) 1214, 1240, l817, 2863, 4102, 4111, 5216, 5443, 5859, 6065, 6100Kpn1 (2) 480, 3895Mae1 (15) 30, 293, 689, 727, 739, 1452, 1606, 1893, 1963, 2483, 3442, 3709, 4975, 5228, 5563Mae2 (11) 808, 1139, 1180, 1987, 2801, 2988, 4233, 5183, 5599, 5972, 6292Mae3 (20) 38, 1052, 1080, 1145, 1289, 1478, 1706, 2805, 3111, 3450, 4134, 4229, 4836, 4899, 5015, 5298, 5629, 5687, 5840, 6028Mbo1 (30) 93, 1102, 1234, 1419, 1657, 2010, 2152, 2521, 2526, 2545, 2856, 2934, 3015, 3024, 3102, 3505, 4019, 5046, 5121, 5132, 5140, 5218, 5230, 5335, 5676, 5694, 5740, 5998, 6015, 6051Mbo2 (17) 444, 1145, 1356, 1575, 1617, 1908, 1911, 3046, 3256, 3336, 4351, 5142, 5213, 5968, 6046, 6155, 6351Mn11 (54) 291, 444, 508, 534, 560, 639, 841, 939, 1227, l330, 1363, 1369, 1372, 1378, 1408, 1411, 1426, 1433, 1449, 1559, 1620, 1909, 1921, 2412, 2418, 2443, 2449, 2455, 2458, 2470, 2508, 2535, 2599, 2735, 3092, 3286, 3707, 3859, 3878, 3923, 3948, 3974, 4054, 4087, 4117, 4379, 4587, 4662, 4911, 5311, 5392, 5540, 5746, 6339Mse1 (22) 35, 1065, 1177, 1207, 1231, 1801, 1843, 1956, 1971, 2124, 2139, 3447, 4261, 5186, 5238, 5243, 5257, 5310, 5545, 5584, 5949, 6321Msp1 (30) 161, 237, 473, 601, 643, 789, 2590, 2667, 2689, 2717, 2848, 2938, 3005, 3186, 3578, 3653, 3888, 4016, 4058, 4126, 4160, 4687, 4834, 4860, 5050, 5454, 5488, 5555, 5665, 5907Mst1 (2) 2787, 5595Mst2 (2) 847, 1076Mva1 (22) 315, 623, 801, 814, 1227, 1252, 1275, 1295, 1325, 1526, 1536, 1558, 1630, 2196, 2251, 2268, 3072, 3731, 4038, 4508, 4629, 4642Nae1 (1) 3187Nar1 (2) 2685, 3860Nci1 (20) 161, 237, 473, 474, 602, 644, 789, 2689, 2849, 3578, 3653, 3888, 3889, 4017, 4059, 4126, 4161, 4860, 5556, 5907Nco1 (2) 2389, 3117Nde1 (1) 4303Nde2 (30) 93, 1102, 1234, 1419, 1657, 2010, 2152, 2521, 2526, 2545, 2856, 2934, 3015, 3024, 3102, 3505, 4019, 5046, 5121, 5132, 5140, 5218, 5230, 5335, 5676, 5694, 5740, 5998, 6015, 6051Nhe1 (3) 29, 1605, 3441Nla3 (26) 61, 1263, 1596, 1649, 1835, 1856, 2030, 2230, 2302, 2393, 2559, 2904, 3090, 3121, 3147, 3473, 4119, 4224, 4484, 5204, 5695, 5705, 5783, 5819, 6212, 6317Nla4 (28) 153, 246, 262, 320, 478, 627, 758, 827, 959, 1202, 1279, 2154, 2200, 2272, 2686, 2721, 3678, 3736, 3861, 3893, 4042, 4512, 4551, 5323, 5417, 5458, 5669, 6259Nsi1 (2) 2232, 2304Nsp(7524)1 (8) 1596, 1835, 1856, 2230, 2302, 3090, 4119, 4484Nsp(7524)2 (19) 323, 413, 426, 597, 1583, 1721, 2631, 2724, 2798, 2988, 3050, 3739, 3828, 3841, 4012, 4300, 4798, 5959, 6044NspB2 (12) 119, 190, 1751, 2158, 2791, 3532, 3607, 3989, 4192, 4822, 5067, 6008NspH1 (8) 1596, 1835, 1856, 2230, 2302, 3090, 4119, 4484PaeR7I (1) 1998Pal1 (35) 171, 202, 247, 658, 792, 828, 840, 1045, 1169, 1190, 1255, 1534, 1650, 1866, 1961, 2423, 2429, 2438, 2481, 2593, 2767, 3158, 3185, 3543, 3588, 3618, 3663, 4495, 4506, 4524, 4958, 5416, 5496, 5763, 6350Ple1 (7) 865, 1547, 3350, 3889, 4374, 4859, 5362PpuM1 (3) 328, 1277, 3744Pss1 (4) 331, 1280, 3747, 6352Pst1 (6) 987, 1163, 1888, 2511, 2738, 5618Pvu1 (1) 5743Pvu2 (6) 119, 190, 1751, 2791, 3532, 3607Rsa1 (10) 347, 478, 725, 1342, 1519, 1597, 2991, 3893, 4288, 5853Rer2 (1) 3201Sac1 (2) 413, 3828Sau1 (2) 847, 1076Sau3A1 (30) 93, 1102, 1234, 1419, 1657, 2010, 2152, 2521, 2526, 2545, 2856, 2934, 3015, 3024, 3102, 3505, 4019, 5046, 5121, 5132, 5140, 5218, 5230, 5335, 5676, 5694, 5740, 5998, 6015, 6051Sau96I (29) 169, 200, 245, 260, 273, 328, 626, 756, 826, 839, 1043, 1254, 1277, 1532, 1649, 3201, 3541, 3586, 3616, 3661, 3676, 3689, 3744, 4041, 5415, 5494, 5511, 5733, 6349Sca1 (1) 5853ScrF1 (42) 161, 237, 315, 473, 474, 602, 623, 644, 789, 801, 814, 1227, 1252, 1275, 1295, 1325, 1526, 1536, 1558, 1630, 2196, 2251, 2268, 2689, 2849, 3072, 3578, 3653, 3731, 3888, 3889, 4017, 4038, 4059, 4126, 4161, 4508, 4629, 4642, 4860, 5556, 5907Sdu1 (19) 323, 413, 426, 597, l583, 1721, 2631, 2724, 2798, 2988, 3050, 3739, 3828, 3841, 4012, 4300, 4798, 5959, 6044Sec1 (38) 159, 235, 314, 324, 472, 536, 621, 622, 760, 799, 800, 812, 813, 1225, 1294, 1303, 1323, 1324, 1525, 1557, 1962, 2194, 2266, 2389, 2424, 2433, 2482, 2848, 3117, 3576, 3651, 3730, 3740, 3887, 3950, 4036, 4037, 4640SfaN1 (23) 258, 520, 997, 1657, 2107, 2239, 2311, 2643, 2898, 2984, 3048, 3114, 3323, 3674, 3934, 4146, 4281, 4317, 4357, 4577, 5629, 5820, 6069Sfi1 (1) 2435Sma1 (2) 474, 3889Spe1 (1) 726Sph1 (4) 1835, 2230, 2302, 3090Ssp1 (1) 6177Sst1 (2) 413, 3828Stu1 (2) 1961, 2481Sty1 (9) 324, 536, 1303, 1962, 2389, 2482, 3117, 3740, 3950Taq1 (15) 860, 1096, 1407, 1418, 1660, 1999, 2514, 2798, 2954, 2978, 3014, 3176, 3367, 4580, 6024Tha1 (19) 392, 394, 445, 969, 971, 1193, 2751, 3052, 3084, 3807, 3809, 4081, 4083, 4186, 4527, 5108, 5438, 5931, 6263Tth111I (6) 465, 877, 1275, 2803, 3880, 4227Xba1 (2) 1892, 3708Xho1 (1) 1998Xho2 (11) 2010, 2152, 2521, 2856, 3102, 5121, 5132, 5218, 5230, 5998, 6015Xma1 (2) 472, 3887Xma3 (2) 790, 2591Xmn1 (1) 5972Xor2 (1) 5743__________________________________________________________________________
TABLE 4______________________________________enzymes which do not cut LXSNRIIL2:______________________________________Acc3 Bg12 Cla1 Hpa1 Nru1 SnaB1Apa1 Bsm1 Dra3 Mlu1 PflM1 Spl1Asu2 BspM2 Eco47III Mro1 Sac2 Sst2Ban3 BstB1 Esp1 Not1 Sal1______________________________________
TABLE 5 - From 1 to 6365. Numbered from position 1. ##STR1## Aat1- - - - - - - - - - - + - - - - - - - - - - - 1 - - - - - 1 - - - - - + - - - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - - - - + - - - - Aat2- - - - - - - - - 1 - + - - - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - - - - + - - 1 - Acc1- - - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - - - - + - - 1 - - - - - - - - + - - - - - - - - - - - + - - - - Acc2- - - - 2 1 - - - - - 2 - - 1 - - - - - - - - + - - - - - - - - 1 - - + 2 - - - - - - - - 2 - + 2 1 - - - 1 - - - - - + 1 - - - 1 - - - - - 1 + - - 1 - Acy1- - - - - - - - - 1 - + - - - - - - - - - - - + - - - - - - - 1 - - - + - - - - - - - - - 1 - + - - - - - - - - - - - + - - - - - - - - - - 1 + - - 1 - Afl1- - - 3 - - - 1 1 - - + - - - 1 - - - - - - - + - - - - - - - - - - - + - - 1 - - - - 2 1 - - + 1 - - - - - - - - - - + - - - - - 1 - - 1 - - + - - - - Afl21 - - - - - - - - - - + 1 - - - - - - - - - - 1 - - - - - - - - - - - + - - - - - 1 - - - - - + - - - - - - - - - - - + - - - - - - - - - - - + - - - - Afl3- - - - - - - - - - - + - - - - - - 1 - - - - + - - - - - - - - - - - + - - - - - - - - - - - + - - - - - 1 - - - - - + - - - - - - - - - - - + - - - - A ha1- 1 1 - - 2 - 2 - 1 - + - - - - - - - - - - - + - - - - - - - - 1 1 - + - - - - - - 1 1 - - 2 1 1 2 - - - - - - - 1 - + - - - - - - 1 - - - 1 + - - - - Aha2- - - - - - - - - 1 - + - - - - - - - - - - - + - - - - - - - 1 - - - + - - - - - - - - - 1 - + - - - - - - - - - - - + - - - - - - - - - - 1 + - - 1 - A ha3- - - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - - - - + - - 2 - - - - - - - 1+ - - - - 2 Alu1 1 1 - 1 - - 1 2 - - + - - - - - 2 - - 1 - - 2 - - - - - 2 - - - 1 - + - - 1 - 1 1 2 - - 1 - + 1 2 - - 1 - - 1 2 - - + 1 - - - - - 1 1 1 - - + - - - - Alw1- - - - - - - - - - - + - 1 - - 1 - - 1 - - - + 1 2 - - - - 2 - - - 2 + - 1 - - - - - - - - - 1 - - - - - - - - - - - + 1 2 2 - - - - 1 - - - 2 - - - - AlwN1- - 1 - - - - - - - - + - - - - - - - - - - - + - - - - - - - - - - - + - - - - - - 1 1 - - - + - - - - - - - - - - 1 + - - - - - - - - - - - + - - - - Aoc1- - - - - - - - - - 1 + 1 - - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - - - - + - - - - A oc2- - - 1 1 1 - 1 - - - + - - - - - - 1 - 1 - - + - - - - - - - 1 1 1 - 1 1 - - - - - - - 1 2 - 1 - - - 1 - - - - - 1 - + - - - - - - - - - - 1 1 - - - - Aos1- - - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - - 1 - + - - - - - - - - - - - + - - - - - - - - - - - + - - - - - - 1 - - - - + - - - - ApaLi- - - - - - - - - - - + - - - - - - - - 1 - - + - - - - - - - - - - - + - - - - - - - - - - - + - - - 1 - - - - - 1 - + - - - - - - - - - - - 1 - - - - Apy1- - - 1 - - - 1 - 2 - + - - 2 3 - - 3 1 - - - + - - 3 - - - - - - - - + 1 - - - - - - - 1 - - + 1 - - - - 1 - 2 - - - + - - - - - - - - - - - + - - - - A qu1- - 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- - - - - - + 1 - - - - - - - - - - + - 1 - - - 1 - - - - - + - - - - - - - - - - - + - - - - PaeR7I- - - - - - - - - - - + - - - - - - - - - - - 1 - - - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - - - - + - - - - Pal1- - 3 - - - - 1 - 3 - + 1 1 2 - - - 1 1 - - 1 1 - - - - 2 2 1 - 1 - - + - 2 - - - - 2 2 - - - + - - - - - 3 - - - - - 1 - - - - 1 1 - - 1 - - + - - - 1 Ple1- - - - - - - - - - 1 + - - - - - - 1 - - - - + - - - - - - - - - - - + - - - 1 - - - - - - 1 + - - - - 1 - - - - 1 - + - - - 1 - - - - - - - + - - - - PpuM1- - - 1 - - - - - - - + - - - 1 - - - - - - - + - - - - - - - - - - - + - - - - - - - - 1 - - + - - - - - - - - - - - + - - - - - - - - - - - + - - - - Pss1- - - 1 - - - - - - - + - - - 1 - - - - - - - + - - - - - - - - - - - + - - - - - - - - 1 - - + - - - - - - - - - - - + - - - - - - - - - - - + - - - 1 Pst1- - - - - - - - - - - 1 - 1 - - - - - - - - 1 + - - - - - 1 - - 1 - - + - - - - - - - - - - - + - - - - - - - - - - - + - - - - - - 1 - - - - + - - - - Pvu1- - - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - 1 - - + - - - - Pvu2- 1 1 - - - - - - - - + - - - - - - - - 1 - - + - - - - - - - - - 1 - + - - - - - - 2 - - - - + - - - - - - - - - - - + - - - - - - - - - - - + - - - - Rsa1- - - - 1 1 - - 1 - - + - - - 1 - - 1 1 - - - + - - - - - - - - - - - 1 - - - - - - - - - - 1 + - - - 1 - - - - - - - + - - - - - - - - - 1 - + - - - - Rsr2- - - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - - - - + - - 1 - - - - - - - - + - - - - - - - - - - - + - - - - - - - - - - - + - - - - Sac1- - - - 1 - - - - - - + - - - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - - 1 - + - - - - - - - - - - - + - - - - - - - - - - - + - - - - S au1- - - - - - - - - - 1 + 1 - - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - - - - + - - - - Sau3A1- 1 - - - - - - - - - + - 1 1 - 1 - - 1 - - - 1 - 1 - - - - 3 - - 1 1 2 1 - - - - 1 - - - - - 1 - - - - - - - - - - - + 2 2 2 1 - - - 2 1 - - 2 1 - - - Sau96I- - 3 3 - - - 1 1 2 - + 1 - 1 1 - - 1 1 - - - + - - - - - - - - - - - + - - 1 - - - 2 4 1 - - + 1 - - - - - - - - - - + - - - - 1 2 - - 1 - - + - - - 1 Sca1- - - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - - 1 - + - - - - ScrF1- 1 1 1 - 2 - 3 - 3 - + - - 2 3 - - 3 1 - - - + - - 3 - - - - - 1 1 - + 1 - - - - - 1 1 1 - 2 1 2 2 - - - 1 - 2 - 1 - + - - - - - - 1 - - - 1 + - - - - Sdu1- - - 1 1 1 - 1 - - - + - - - - - - 1 - 1 - - + - - - - - - - 1 1 1 - 1 1 - - - - - - - 1 2 - 1 - - - 1 - - - - - 1 - + - - - - - - - - - - 1 1 - - - - Sec1- 1 1 2 - 1 1 2 - 5 - + - - 1 4 - - 2 - - - - 1 - - 2 - 3 1 - - - 1 - + - 1 - - - - 1 1 2 - 1 1 2 - - - - - - 1 - - - + - - - - - - - - - - - + - - - - S faN1- - - 1 - - 1 - - - - 1 - - - - - - - 1 - - - + - 1 1 1 - - - 1 - - 1 1 1 1 - 1 - - - 1 - - 1 + - 1 1 2 - - 1 - - - - + - - - - - - - 1 - 1 - + 1 - - - Sfi1- - - - - - - - - - - + - - - - - - - - - - - + - - - - 1 - - - - - - + - - - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - - - - + - - - - S ma1- - - - - 1 - - - - - + - - - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - - - 1 + - - - - - - - - - - - + - - - - - - - - - - - + - - - - Spe1- - - - - - - - 1 - - + - - - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - - - - + - - - - Sph1- - - - - - - - - - - + - - - - - - - - - 1 - + - - 1 1 - - - - - - - + 1 - - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - - - - + - - - - Ssp1- - - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - - - - + - 1 - - S st1- - - - 1 - - - - - - + - - - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - - 1 - + - - - - - - - - - - - + - - - - - - - - - - - + - - - - Stu1- - - - - - - - - - - + - - - - - - - - - - - 1 - - - - - 1 - - - - - + - - - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - - - - + - - - - Sty1- - - 1 - - 1 - - - - + - - - 1 - - - - - - - 1 - - - - 1 1 - - - - - + - 1 - - - - - - 1 - - 1 - - - - - - - - - - - + - - - - - - - - - - - + - - - - Tag1- - - - - - - - - - 1 + - 1 - - 2 - - 1 - - - 1 - - - - - 1 - - - 1 - 3 - 1 - - 1 - - - - - - + - - - - - - 1 - - - - + - - - - - - - - - - - 1 - - - - T ha1- - - - 2 1 - - - - - 2 - - 1 - - - - - - - - + - - - - - - - - 1 - - + 2 - - - - - - - - 2 - + 2 1 - - - 1 - - - - - + 1 - - - 1 - - - - - 1 + - - 1 - Tthl11I- - - - - 1 - - - - 1 + - - - 1 - - - - - - - + - - - - - - - - - 1 - + - - - - - - - - - - 1 + - - 1 - - - - - - - - + - - - - - - - - - - - + - - - - Xba1- - - - - - - - - - - + - - - - - - - - - - 1 + - - - - - - - - - - - + - - - - - - - - 1 - - + - - - - - - - - - - - + - - - - - - - - - - - + - - - - Xho1- - - - - - - - - - - + - - - - - - - - - - - 1 - - - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - - - - + - - - - Xho2- - - - - - - - - - - + - - - - - - - - - - - 1 - 1 - - - - 1 - - 1 - + 1 - - - - - - - - - - + - - - - - - - - - - - + 1 1 2 - - - - - - - - 2 - - - - X ma1- - - - - 1 - - - - - + - - - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - - - 1 + - - - - - - - - - - - + - - - - - - - - - - - + - - - - Xma3- - - - - - - - - 1 - + - - - - - - - - - - - + - - - - - - 1 - - - - + - - - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - - - - + - - - - Xmn1- - - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - - - - 1 - - - - Xor2- - - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - - - - + - - - - - - - - 1 - - + - - - -
TABLE 6__________________________________________________________________________from 1 to 6365. Numbered from position 1. ##STR2## ##STR3## ##STR4## ##STR5## ##STR6## ##STR7## ##STR8## ##STR9## ##STR10## ##STR11## ##STR12## ##STR13## ##STR14## ##STR15## ##STR16## ##STR17## ##STR18## ##STR19## ##STR20## ##STR21## ##STR22## ##STR23## ##STR24## ##STR25## ##STR26## ##STR27## ##STR28## ##STR29## ##STR30## ##STR31## ##STR32## ##STR33## ##STR34## ##STR35## ##STR36## ##STR37## ##STR38## ##STR39## ##STR40## ##STR41## ##STR42## ##STR43## ##STR44## ##STR45## ##STR46## ##STR47## ##STR48## ##STR49## ##STR50## ##STR51## ##STR52## ##STR53## ##STR54## ##STR55## ##STR56## ##STR57## ##STR58## ##STR59## ##STR60## ##STR61## ##STR62## ##STR63## ##STR64## ##STR65## ##STR66## ##STR67## ##STR68## ##STR69## ##STR70## ##STR71## ##STR72## ##STR73## ##STR74## ##STR75## ##STR76## ##STR77## ##STR78## ##STR79## ##STR80## ##STR81## ##STR82## ##STR83## ##STR84## ##STR85## ##STR86## ##STR87## ##STR88## ##STR89## ##STR90## ##STR91## ##STR92## ##STR93## ##STR94##__________________________________________________________________________ zymes which do not cut LXSNRIIL2: - ##STR95##
To generate the LXSN-RI-IL2 retroviral vector, 10 micrograms of pLXSN-RI-IL2 DNA was transfected into the ecotropic packaging cell line PE501 by standard calcium phosphate precipitation methods (Miller et al., Mol. Cell Biol. 6:2895, 1986). The transfected PE501 cell line was grown in DMEM medium with 10% FCS. The medium was changed after 24 hours and supernatant harvested 24 hours later to infect the amphotropic packaging cell line PA317 as described (Miller et al., Mol. Cell Biol. 6:2895, 1986 and Miller et al., BioTechniques 7:980, 1989). The infected PA317 cells were harvested by trypsinization 24 hours later and replated 1:20 in DMEM containing 10% FCS and the neomycin analogue G418 (400 .mu.g/ml). The cells were grown at 37.degree. C. in 7% CO.sub.2 atmosphere. The selection medium was changed every 5 days until colonies appeared. On day 14, twenty colonies were selected, expanded and tested for vital production by standard methods (Xu et al., Virology 171:331-341, 1989). Briefly, supernatants were harvested from confluent culture dishes, passed through a 0.45 .mu.m filter, diluted with DMEM with 10% FCS and utilized to infect NIH 3T3 cells in the presence of 8 .mu.g/ml polybrene. After 24 hours, the infected NIH 3T3 cells were grown in culture medium that contained the neomycin analogue G418. After 12-14 days, the colonies were stained, counted and the viral titer calculated as described (Xu et al., Virology 171:331-341, 1989).
Colonies with the highest viral titers (>10.sup.4 infectious units/ml) were tested for IL-2 expression by Northern blot analyses. Colonies with the highest viral titers and documented IL-2 expression were cryopreserved and will be utilized as stock cultures to produce the LXSN-RI-IL2 retroviral vector trial.
EXAMPLE IV
Retroviral Vector Construction and Cytokine Expression
To increase IL-2 production by transduced cell lines, vectors were used containing different promoters to drive IL-2 expression, and a human IL-2 cDNA was directionally sub-cloned into the insulin secretory signal peptide (17). The IL-2 cDNA was directionally sub-cloned into the parental plasmids of the LXSN (LTR promoter) and LNCX (CMV promoter) vectors (gifts of Dr. A. D. Miller) (18). The newly constructed vectors (FIG. 1), designated as LXSN-IL2 and LNCX-IL2, were packaged in the PA317 cell line for production of retroviral supernatant. As a control, the high level expressing, double copy vector DC/TKIL-2 vector (thymidine kinase promoter) (a gift of Dr. E. Gilboa) was used for comparison.
These vectors were used to transduce a number of murine and human, primary and established cell lines. Pools of transduced cells were selected and expanded in DMEM medium, containing 10% fetal bovine serum (FBS) and 400 .mu.g/ml of active G-418, a neomycin analogue. The results of expression studies in the MCR9 and Balb/c 3T3 cell lines are presented in Table 7.
TABLE 7______________________________________Comparison of IL-2 expression by fibroblaststransduced with different IL-2 vectors. ng IL-2 Units IL-2Fibroblast Vector per 10.sup.6 cells per day______________________________________Murine LNCX (Control) 0.4 .+-. 50% <1 LNCX-IL2 33.7 .+-. 11% 67 LXSN-IL2 6.6 .+-. 6% 13 DC/TKIL-2 1.9 .+-. 5% 4Human LXSN (Control) 0.7 .+-. 29% 1 LNCX-IL-2 159.5 .+-. 17% 319 LXSN-IL2 25.5 .+-. 15% 51 DC/TKIL-2 3.0 .+-. 10% 6______________________________________
EXAMPLE V
Fibroblast Culture and Conditions for Retroviral Transduction
The culture conditions for the growth of primary fibroblasts retroviral transduction were optimized. Primary fibroblasts were successfully cultured. The optimal conditions enable the growth of approximately 3-4.times.10.sup.6 primary fibroblasts from a 12 mm.sup.2 skin biopsy in approximately 4-6 weeks. Retroviral infection, G418 selection, and expansion of the genetically modified fibroblasts takes an additional 4-6 weeks.
Exploring the conditions for genetic modification of primary fibroblasts suggests that optimal transduction may be obtained by the following procedure: The fibroblasts are synchronized in G1 phase by serum starvation, followed by stimulation with medium containing 15% fetal bovine serum 15 hours prior to transduction. The cells are then subjected to 2 cycles of retrovirus infection, each cycle lasting approximately 3 hours. The cells are refed with fresh media overnight, and then selection in G418 is initiated the next day. This method is capable of transducing 5-15% of the fibroblasts in a culture, depending on the multiplicity of infection.
This procedure was used to transduce a large number of primary and established fibroblasts. As an example, Table 8 compares the expression levels of IL-2 in fibroblast lines transduced with LXSN-IL2.
TABLE 8______________________________________Expression of IL-2 by fibroblasts transduced with LXSN-IL2.Fibroblast ng IL-2 Units IL-2Line Species Origin per 10.sup.6 cells per day______________________________________Balb/c 3T3 Murine Transformed 6.6 .+-. 6% 13MCR9 Human Embryonic 25.5 .+-. 15% 51NHDF 313 Human Skin 25.0 .+-. 10% 50GT1 Human Skin 15.0 .+-. 5% 30______________________________________
These results indicate that the IL-2 expression levels in established, embryonic, and primary fibroblast cultures are similar. Comparison of these data with Table 7 suggest that IL-2 expression is affected more by factors such as different promoters than by the fibroblast line used. Similarly, changes in culture conditions can have important effects on IL-2 expression. Table 9 shows that transduced GT1 cells, a primary human fibroblast culture expressed 15-fold more IL-2 under 100 .mu.g/ml G418 selection than under 25 .mu.g/ml G418 selection. Several other primary fibroblast lines have also been transduced with our vectors and are currently growing under G418 selection.
TABLE 9______________________________________Effect of G418 concentration on IL-2 expression by GT1cells transduced with LXSN-IL2.Selection dose ng IL-2 secretedof G418 per 10.sup.6 cells per day*______________________________________25 .mu.g/ml 1.0 .+-. 10%50 .mu.g/ml 3.0 .+-. 6%100 .mu.g/ml 15.0 .+-. 5%______________________________________ *After three weeks of G418 selection.
EXAMPLE VI
Comparison of IL-2 Expression Levels Induced Peripheral Blood Lymphocytes and Genetically Modified Fibroblasts
In order to compare the production of IL-2 by genetically modified fibroblasts to that achieved by stimulating normal human peripheral blood lymphocytes (nPBL) in vitro, nPBL were isolated by Ficol-Paque density centrifugation, and cultured in the presence of allogeneic nPBL (mixed lymphocyte culture, MLC) or 2 .mu.M calcium ionophore (CI) (A23187) free acid) plus 17 nM phorbol 12-myristate 13-acetate (PMA). The results of this experiment, present in Table 10, indicate that the level of IL-2 expression in the PMA/CI stimulated normal T cell population was 2 ng/10.sup.6 cells/24 hours. This is equivalent to IL-2 expression by Balb/c 3T3 fibroblasts transduced with DC/TKIL-2 (Table 7), our least productive vector. The level of IL-2 expression in the MLC was 130 pg/10.sup.6 cells/24 hours. This was lower than the PMA/CI stimulated culture, presumably because PMA/CI induced a nonspecific response while MLC resulted in specific Th stimulation. When the estimated percentage of antigen-specific Th in the MLC-stimulated population is taken into consideration, the level of IL-2 expression per stimulated T cell becomes equivalent for both methods.
TABLE 10______________________________________Levels of IL-2 secretion by different cells. pg IL-2 secretedCells per 10.sup.6 cells per day______________________________________Lymphocytes:Control (non-activated) 5 .+-. 50%PMA + Calcium Ionophore 2,000 .+-. 6%Mixed lymphocyte culture 130 .+-. 90%Transduced fibroblasts:MCR9-LXSN-IL2 24,000 .+-. 5%MCR9-LNCX-IL2 162,000 .+-. 20%MCR9-DC/TKIL-2 10,000 .+-. 6%______________________________________
EXAMPLE VII
Fibroblast Mediated Cytokine Gene Therapy in Murine Tumor Models
Two experimental protocols were used to study the efficacy of fibroblast-mediated cytokine gene therapy on induction of anti-tumor immunity. The first protocol was designed to test the effects of genetically modified fibroblasts on tumor implantation, while the second protocol was designed to induce a systemic anti-tumor immunity. The results of each experiment are presented with two figures and one table. In the first figure, the rate of tumor growth for each treatment group is presented as the mean tumor size in the group over time. In the second figure, a Kaplan-Meier curve presents the time of tumor onset for the individual animals in each treatment group. The number of animals, the number and percentage of tumor free animals, and the tumor size distribution patterns for each experiment are presented in a table.
EXAMPLE VII
Effect of Fibroblast Mediated Cytokine Gene Therapy on Tumor Implantation
Mice were injected subcutaneously with mixtures of 5.times.10.sup.4 CT26 cells and 2.times.10.sup.6 fibroblasts genetically modified by different retroviral vectors to express IL-2. In the control arms injected with tumor cells only, or with tumor cells mixed with unmodified fibroblasts, 31 of 33 animals (94%) developed tumors by 4 weeks (FIGS. 6 and 7, Table 9). In contrast, 22 out of the 34 animals (65%) receiving fibroblast mediated cytokine gene therapy were tumor free at 3 weeks, and 5 animals (18%) remain tumor free after 12 weeks. Those animals that received fibroblast mediated IL-2 therapy and developed tumor were characterized by a delayed onset and rate of tumor growth.
TABLE 11__________________________________________________________________________Effect of IL-2 modified fibroblasts on tumor establishment anddevelopment.2 .times. 10.sup.6 fibroblasts mixed with 5 .times. 10.sup.4 CT26 tumorcells at time of injection. Animal NumberFibroblasts Tumor- Tumor- Percent Tumor Size (mm.sup.2) Median Tumor Sizemixed with tumor cells Total free bearing Tumor-free 25-100 101-200 201-300 >301 (mm.sup.2)__________________________________________________________________________After 12 Weeks:*Control (no fibroblasts) 11 0 11 0% 1 0 1 9 420 .+-. 145Unmodified fibroblasts** 13 2 11 15% 1 0 1 7 389 .+-. 265DCTK-IL2 fibroblasts 13 0 13 0% 1 3 5 4 267 .+-. 168LNCX-IL2 fibroblasts 13 5 8 39% 5 2 0 1 72 .+-. 90__________________________________________________________________________ * Mean tumor size is for 4 weeks, the last timepoint at which tumors were measured. **Two mice in this arm developed intraperitoneal tumors which were not measurable.
After 3 weeks the mean tumor size (measured as the product of the longest and widest tumor axes) in the control group of mice was 128 mm.sup.2, compared to 68 and 7 mm.sup.2 in groups of mice injected with tumor cells mixed with fibroblasts transduced with DC/TKIL-2 or LNCX-IL2, respectively. This resulted in a highly significant difference (corrected x.sup.2 =18.69, p=0.001) between the IL-2 treated animals compared to the mice treated with CT26 alone or CT26 mixed with unmodified fibroblasts. After four weeks the equivalent measurements were 373,300 and 72 mm.sup.2 (Table 11). It is notable that LNCX-IL2, the highest expressing vector caused substantially greater inhibition of tumorigenicity than the lower expressing vector DC/TKIL-2. A multivariate non-parametric statistical procedure (19,20), utilized to evaluate differences in tumor growth, demonstrated that after 4 weeks the differences between the growth curves for the four groups presented in FIG. 2 were highly significant (p<0.001). Subsequent comparisons between the control arm and animals that received tumor cells mixed with IL-2 transduced fibroblasts revealed a significant difference (P<0.05). The differences between the animals injected with tumor cells alone, and those injected with tumor cells plus unmodified fibroblasts were not significant, while the differences between animals receiving low IL-2 expressing fibroblast, and those receiving high IL-2 expressing fibroblasts was significant (P=0.05).
When mice were injected with 2.times.10.sup.6 modified fibroblasts mixed with 1.times.10.sup.5 live tumor cells the results became more striking (see FIGS. 8 and 9, and Table 12). All the control animals developed tumors after 4 weeks whereas 33% and 27% of the animals treated with fibroblasts modified with the DCTK-IL2 or LXSN-IL2 vectors (respectively) remain tumor free after 7 weeks (the experiment is ongoing). More dramatically, 75% of the animals treated with fibroblasts modified with the highest IL-2 producing vector, LNCX-IL2, remain tumor free after 7 weeks. These data clearly demonstrate the importance of an initial high dose of IL-2 to prevent tumor establishment.
TABLE 12__________________________________________________________________________Effect of IL-2 modified fibroblasts on tumor establishment anddevelopment.2 .times. 10.sup.6 fibroblasts mixed with 1 .times. 10.sup.5 CT26 tumorcells at time of injection. Animal NumberFibroblasts Tumor- Tumor- Percent Tumor Size (mm.sup.2) Mean Tumor Sizemixed with tumor cells Total free bearing Tumor-free 25-100 101-200 201-300 >301 (mm.sup.2)__________________________________________________________________________After 6 Weeks:*Control (no fibroblasts)** 13 0 13 0% 0 5 2 5 315 .+-. 197Unmodified fibroblasts** 20 0 20 0% 0 2 3 14 350 .+-. 100DCTK-IL2 fibroblasts 12 4 8 33% 0 1 4 3 185 .+-. 141LXSN-IL2 fibroblasts*** 15 4 11 27% 0 5 1 2 135 .+-. 121LNCX-IL2 fibroblasts 8 6 2 75% 2 0 0 0 8 .+-. 14__________________________________________________________________________ * Mean tumor size is for 4 weeks, the last timepoint at which tumors were measured. **One mouse in each of these arms developed an intraperitoneal tumor whic was not measurable. ***Three mice in this arm developed intraperitoneal tumors which were not measurable.
As an additional control, mice were injected with CT26 cells genetically modified to express IL-2 (results not shown). Injection of up to 1.times.10.sup.6 IL-2 expressing tumor cells into Balb/c mice failed to produce tumors. Injection of higher numbers however, resulted in some animals developing tumors with delayed onset. These data confirm the results reported in the literature (1). In order to compare the efficacy of IL-2 producing fibroblasts to IL-2 producing tumor cells, we mixed 2.times.10.sup.6 CT26 tumor cells modified with the DCTK-IL2 vector with 1.times.10.sup.5 unmodified tumor cells. FIGS. 10 and 11, and Table 13 show that DCTK-IL2 modified tumor cells are somewhat effective in preventing tumor development. Four weeks after injection, the mean tumor size for the treatment arm is 303 mm.sup.2, compared to 620 mm.sup.2 for the control arm. After 22 weeks, one animal (10%) remains tumor free, compared to none in the control arms. Data for animals treated under the same conditions with DCTK-IL2 modified fibroblasts in a separate experiment are included for comparison purposes. This comparison suggests that DCTK-IL2 modified tumor cells have an effect on tumor establishment similar to that of DCTK-IL2 modified fibroblasts.
TABLE 13__________________________________________________________________________Effect of IL-2 modified cells on tumor establishment and developement.2 .times. 10.sup.6 DCTK-IL2-modified CT26 tumor cells mixed with 1.times. 10.sup.5 CT26 cells compared to 2 .times. 10.sup.6DCTK-IL2-modified fibroblasts mixed with 1 .times. 10.sup.5 CT26. Animal NumberCells Tumor- Tumor- Percent Tumor Size (mm.sup.2) Mean Tumor Sizemixed with tumor cells Total free bearing Tumor-free 25-100 101-200 201-300 >301 (mm.sup.2)__________________________________________________________________________After 22 Weeks:*Control (no fibroblasts) 5 0 5 0% 0 0 0 5 620 .+-. 190Unmodified fibroblasts 5 0 5 0% 0 0 0 5 587 .+-. 69DCTK-IL2-modified 10 1 9 10% 1 0 2 5 303 .+-. 179CT26 cellsDCTK-IL2-modified 8 2 6 25% 0 1 2 3 214 .+-. 158fibroblasts__________________________________________________________________________ *Mean tumor size is for 4 weeks, the last timepoint at which tumors were measured.
EXAMPLE VII(b)
Effect of Fibroblast Mediate Cytokine Gene Therapy on Systemic Anti-tumor Immunity
Groups of Balb/c mice were immunized with 2.5.times.10.sup.5 irradiated tumor cells either alone or mixed with 2.times.10.sup.6 transduced or unmodified fibroblasts, and challenged one week later with 5.times.10.sup.4 live tumor cells in the opposite flank. These results (FIGS. 12 and 13, and Table 14) demonstrate that immunization with irradiated tumor cells and transduced fibroblasts protect some animals against a live tumor challenge, but that the protection is only slightly better than that achieved by immunization with irradiated tumor cells alone or irradiated tumor cells mixed with unmodified fibroblasts.
TABLE 14__________________________________________________________________________Effect of Il-2 modified fibroblasts on induction of sytemic anti-tumorimmunity.Mice immunized with 2 .times. 10.sup.6 fibroblasts mixed with 2.5 .times.10.sup.5irradiated CT26 tumor cells 7 days prior to challenge with 5 .times.10.sup.4 fresh tumor cells. Animal NumberFibroblasts mixed with Tumor- Tumor- Percent Tumor Size (mm.sup.2) Mean Tumor Sizeirradiated tumor cells Total free bearing Tumor-free 25-100 101-200 201-300 >301 (mm.sup.2)__________________________________________________________________________After 22 Weeks:*Control (saline) 20 0 20 0% 0 0 1 19 574 .+-. 160Irradiated CT26 only** 16 5 11 31% 2 1 2 5 250 .+-. 277Irradiated CT26 mixed with 15 4 11 27% 0 1 3 7 266 .+-. 199unmodified fibroblastsDCTK-IL2 fibroblasts** 25 10 15 40% 4 1 1 8 172 .+-. 194__________________________________________________________________________ *Mean tumor size is for 4 weeks, the last timepoint at which tumors were measured. ** One mouse in each of these arms developed an intraperitoneal tumor which was not measurable.
In a second protocol similar to the one described above, animals were challenged with fresh tumor cells two weeks following immunization with irradiated tumor cells mixed with fibroblasts. The results, shown in FIGS. 14 and 15, and in Table 15, demonstrate that DCTK-IL2 modified fibroblasts mixed with irradiated tumor cells confers superior protection to subsequent tumor challenge than irradiated tumor cells alone, irradiated tumor cells mixed with unmodified fibroblasts, or irradiated tumor cells mixed with LNCX-modified fibroblasts. After 7 weeks, seven of ten animals (70%) treated with DCTK-IL2 modified fibroblasts remain tumor free compared to only one third of the control animals. At four weeks, the mean tumor size of this group was 41 mm.sup.2, compared to 180, 170, and 140 mm.sup.2 for the three control groups. Animals treated with LNCX-IL2 modified fibroblasts were also protected against subsequent tumor challenge, but the results were less striking. In this group, 54% of the animals remain tumor free and the mean tumor size for the group at four weeks was 86 mm.sup.2. The number of tumor free animals in the group treated with LXSN-IL2 modified fibroblasts was similar to the control groups, although the tumors were slightly delayed in their onset. A multivariate non-parametric statistical procedure (19, 20), utilized to evaluate differences in tumor onset, demonstrated that the differences for the six arms presented in FIG. 15 were significant (p=0.012). It further showed that the saline control arm and the arms that received irradiated tumor cells alone or mixed with unmodified or LNCX vector modified fibroblasts formed a statistical group. A second, distinct statistical group was formed by the three arms that received IL-2 vector modified fibroblasts mixed with irradiated tumor cells. Subsequent comparisons between the saline injected control arm and animals that received tumor cells mixed with IL2 transduced fibroblasts revealed a significant difference for all vectors (p<0.05).
TABLE 15__________________________________________________________________________Effect of IL-2 modified fibroblasts on induction of sytemic anti-tumorimmunity.Mice immunized with 2 .times. 10.sup.6 fibroblasts mixed with 2.5 .times.10.sup.5irradiated CT26 tumor cells 14 days prior to challenge with 5 .times.10.sup.4 fresh tumor cells.Immunization by Animal Numberfibroblasts mixed with Tumor- Tumor- Percent Tumor Size (mm.sup.2) Mean Tumor Sizeirradiated tumor cells Total free bearing Tumor-free 25-100 101-200 201-300 >301 (mm.sup.2)__________________________________________________________________________After 7 Weeks:*Control (saline)** 8 1 7 13% 0 2 1 3 245 .+-. 173Irradiated CT26 only 10 3 7 30% 0 2 4 1 180 .+-. 155Irradiated CT26 mixed with 6 2 4 33% 0 2 1 1 170 .+-. 160unmodified fibroblastsIrradiated CT26 mixed with 10 3 7 30% 3 0 1 3 140 .+-. 142LNCX-modified fibroblastsIrradiated CT26 mixed with 13 7 6 54% 1 3 1 1 86 .+-. 112LNCX-IL2-modified fibroblastsIrradiated CT26 mixed with 12 4 8 33% 5 0 2 1 111 .+-. 145LXSN-IL2-modified fibroblastsIrradiated CT26 mixed with 10 7 3 70% 1 2 0 0 41 .+-. 75DCTX-IL2-modified fibroblasts__________________________________________________________________________ *Mean tumor size is for 4 weeks, the last timepoint at which tumors were measured. **One mouse in this arm developed an intraperitoneal tumor which was not measurable.
These results demonstrate the feasibility of using genetically modified fibroblasts as a means of delivering cytokine gene therapy. In all experiments, the LNCX-L2 vector proved superior in preventing tumor establishment while the DCTK-IL2 vector was better in the induction of systemic protection against subsequent tumor challenges. These contrasting effects, although somewhat surprising, can be explained by the observation that the CMV promoter is turned off in vivo five days after implantation while the TK promoter remains active for a longer period of time. The implication of this finding is that to apply this method of gene therapy successfully we have to use promoters that result in high level, sustained expression of IL-2 in vivo in the transduced fibroblasts.
The data obtained from this research effort has important implications for all cytokines that have either direct or indirect anti-tumor effects. Furthermore, this data suggests that anti-tumor efficacy is IL-2 dose dependent. Hence, construction of vectors which result in higher levels of cytokine secretion will be a significant advance toward the application of this method of gene therapy.
Reference numbers in parenthesis in the above examples correspond to the following list of references and are incorporated herein by reference.
References
1. Gabrilove, J. L. et al., Monogr. J. Natl. Cancer Inst. 10:73-7 (1990).
2. Kelso, A., Current Opinion in Immunology, 2:215-25 (1989).
3. Borden, E. C. et al., Cancer, 65:800-14 (1990).
4. Rosenberg, S. A. et al., Ann. Intern. Med., 108:853-864 (1988).
5. Lotze, M. T. et al., JAMA, 256:3117-3124 (1986).
6. Pizza, G. et al., Lymphokine Research, 7:45-8 (1988).
7. Sarna, G. et al., Journal of Biological Response Modifiers, 9:81-6 (1990).
8. Gandolfi, L. et al., Hepato-Gastroenterology, 36:352-6 (1989).
9. Bubenik, J. et al., Immunol. Letters, 19:279-82 (1988).
10. Bubenik et al., Immunol. Letters, 23:287-292 (1990).
11. Fearon, E. R. et al., Cell, 60:387-403 (1990).
12. Gansbacher, B. et al., J. Exp. Med., 172:1217-1224 (1990).
13. Watanabe, Y. et al., Proc. Natl. Acad. Sci., 86:9456-9460 (1989).
14. Tepper, R. I. et al., Cell, 57:503-512 (1989).
15. Kriegler, M., Gene Transfer and Expression: A Laboratory Manual, Stockton Press (1990).
16. Rosenberg, S. A. et al., N. Eng. J. Med., 370 (1990).
17. Cornetta, K. et al., Prog. Nucl. Acid Res. Mol. Biol., 36:311-22 (1989).
18. Hoover, H. C. et al., Cancer Res., 44:1671-76 (1984).
19. Sobol et al. New Eng. J. Med. 316:1111-1117 (1987).
20. Li Xu, et al., Virology, 171:331-341 (1989).
Although the invention has been described with reference to the presently-preferred embodiment, it should be understood that various modifications can be made without departing from the spirit of the invention. Accordingly, the invention is limited only by the following claims.
__________________________________________________________________________SEQUENCE LISTING(1) GENERAL INFORMATION:(iii) NUMBER OF SEQUENCES: 2(2) INFORMATION FOR SEQ ID NO:1:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 6365 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: double(D) TOPOLOGY: linear(ii) MOLECULE TYPE: DNA (genomic)(iii) HYPOTHETICAL: NO(iv) ANTI-SENSE: NO(ix) FEATURE:(A) NAME/KEY: misc.sub.-- feature(B) LOCATION: complement (1..6365)(D) OTHER INFORMATION: /note= "Complementary strand ofpLXSN-RI- IL2"(ix) FEATURE:(A) NAME/KEY: CDS(B) LOCATION: 2557..3351(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:TTTGAAAGACCCCACCCGTAGGTGGCAAGCTAGCTTAAGTAACGCCACTTTGCAAGGCAT60GGAAAAATACATAACTGAGAATAGGAAAGTTCAGATCAAGGTCAGGAACAAAGAAACAGC120TGAATACCAAACAGGATATCTGTGGTAAGCGGTTCCTGCCCCGGCTCAGGGCCAAGAACA180GATGAGACAGCTGAGTGATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGG240CTCGGGGCCAAGAACAGATGGTCCCCAGATGCGGTCCAGCCCTCAGCAGTTTCTAGTGAA300TCATCAGATGTTTCCAGGGTGCCCCAAGGACCTGAAAATGACCCTGTACCTTATTTGAAC360TAACCAATCAGTTCGCTTCTCGCTTCTGTTCGCGCGCTTCCGCTCTCCGAGCTCAATAAA420AGAGCCCACAACCCCTCACTCGGCGCGCCAGTCTTCCGATAGACTGCGTCGCCCGGGTAC480CCGTATTCCCAATAAAGCCTCTTGCTGTTTGCATCCGAATCGTGGTCTCGCTGTTCCTTG540GGAGGGTCTCCTCTGAGTGATTGACTACCCACGACGGGGGTCTTTCATTTGGGGGCTCGT600CCGGGATTTGGAGACCCCTGCCCAGGGACCACCGACCCACCACCGGGAGGTAAGCTGGCC660AGCAACTTATCTGTGTCTGTCCGATTGTCTAGTGTCTATGTTTGATGTTATGCGCCTGCG720TCTGTACTAGTTAGCTAACTAGCTCTGTATCTGGCGGACCCGTGGTGGAACTGACGAGTT780CTGAACACCCGGCCGCAACCCTGGGAGACGTCCCAGGGACTTTGGGGGCCGTTTTTGTGG840CCCGACCTGAGGAAGGGAGTCGATGTGGAATCCGACCCCGTCAGGATATGTGGTTCTGGT900AGGAGACGAGAACCTAAAACAGTTCCCGCCTCCGTCTGAATTTTTGCTTTCGGTTTGGAA960CCGAAGCCGCGCGTCTTGTCTGCTGCAGCATCGTTCTGTGTTGTCTCTGTCTGACTGTGT1020TTCTGTATTTGTCTGAAAATTAGGGCCAGACTGTTACCACTCCCTTAAGTTTGACCTTAG1080GTCACTGGAAAGATGTCGAGCGGATCGCTCACAACCAGTCGGTAGATGTCAAGAAGAGAC1140GTTGGGTTACCTTCTGCTCTGCAGAATGGCCAACCTTTAACGTCGGATGGCCGCGAGACG1200GCACCTTTAACCGAGACCTCATCACCCAGGTTAAGATCAAGGTCTTTTCACCTGGCCCGC1260ATGGACACCCAGACCAGGTCCCCTACATCGTGACCTGGGAAGCCTTGGCTTTTGACCCCC1320CTCCCTGGGTCAAGCCCTTTGTACACCCTAAGCCTCCGCCTCCTCTTCCTCCATCCGCCC1380CGTCTCTCCCCCTTGAACCTCCTCGTTCGACCCCGCCTCGATCCTCCCTTTATCCAGCCC1440TCACTCCTTCTCTAGGCGGGAATTCGTTAGCTTGGTAAGTGACCAGCTACAGTCGGAAAC1500CATCAGCAAGCAGGTATGTACTCTCCAGGGTGGGCCTGGCTTCCCCAGTCAAGACTCCAG1560GGATTTGAGGGACGCTGTGGGCTCTTCTCTTACATGTACCTTTTGCTAGCCTCAACCCTG1620ACTATCTTCCAGGTCATTGTTCCAACATGGCCCTGTGGATCGACAGGATGCAACTCCTGT1680CTTGCATTGCACTAAGTCTTGCACTTGTCACAAACAGTGCACCTACTTCAAGTTCTACAA1740AGAAAACACAGCTGCAACTGGAGCATTTACTGCTGGATTTACAGATGATTTTGAATGGAA1800TTAATAATTACAAGAATCCCAAACTCACCCGCATGCTCACATTTAAGTTTTACATGCCCA1860AGAAGGCCACAGAACTGAAACATCTGCAGTGTCTAGAAGAAGAACTCAAACCTCTGGAGG1920AAGTGCTAAATTTAGCTCAAAGCAAAAACTTTCACTTAAGGCCTAGGGACTTAATCAGCA1980ATATCAACGTAATAGTTCTCGAGCTAAAGGGATCTGAAACAACATTCATGTGTGAATATG2040CTGATGAGACAGCCACCATTGTGGAATTTCTGAACAGATGGATTACCTTTTGTCAAAGCA2100TCATCTCAACACTAACTTGATAATTAAGTGCTTCCCACTTAAAACATATCAGGATCCGCT2160GTGGAATGTGTGTCAGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTAT2220GCAAAGCATGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGC2280AGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAAC2340TCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACT2400AATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTA2460GTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTTGGGCTGCAGGTCGAGGC2520GGATCTGATCAAGAGACAGGATGAGGATCGTTTCGCATGATTGAACAAGATGGA2574MetIleGluGlnAspGly15TTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTAT2622LeuHisAlaGlySerProAlaAlaTrpValGluArgLeuPheGlyTyr101520GACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGG2670AspTrpAlaGlnGlnThrIleGlyCysSerAspAlaAlaValPheArg253035CTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCC2718LeuSerAlaGlnGlyArgProValLeuPheValLysThrAspLeuSer404550GGTGCCCTGAATGAACTGCAGGACGAGGCAGCGCGGCTATCGTGGCTG2766GlyAlaLeuAsnGluLeuGlnAspGluAlaAlaArgLeuSerTrpLeu55606570GCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAA2814AlaThrThrGlyValProCysAlaAlaValLeuAspValValThrGlu758085GCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTC2862AlaGlyArgAspTrpLeuLeuLeuGlyGluValProGlyGlnAspLeu9095100CTGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGAT2910LeuSerSerHisLeuAlaProAlaGluLysValSerIleMetAlaAsp105110115GCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGAC2958AlaMetArgArgLeuHisThrLeuAspProAlaThrCysProPheAsp120125130CACCAAGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCC3006HisGlnAlaLysHisArgIleGluArgAlaArgThrArgMetGluAla135140145150GGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAGGGGCTCGCG3054GlyLeuValAspGlnAspAspLeuAspGluGluHisGlnGlyLeuAla155160165CCAGCCGAACTGTTCGCCAGGCTCAAGGCGCGCATGCCCGACGGCGAG3102ProAlaGluLeuPheAlaArgLeuLysAlaArgMetProAspGlyGlu170175180GATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCATGGTG3150AspLeuValValThrHisGlyAspAlaCysLeuProAsnIleMetVal185190195GAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTG3198GluAsnGlyArgPheSerGlyPheIleAspCysGlyArgLeuGlyVal200205210GCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAA3246AlaAspArgTyrGlnAspIleAlaLeuAlaThrArgAspIleAlaGlu215220225230GAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTATC3294GluLeuGlyGlyGluTrpAlaAspArgPheLeuValLeuTyrGlyIle235240245GCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAG3342AlaAlaProAspSerGlnArgIleAlaPheTyrArgLeuLeuAspGlu250255260TTCTTCTGAGCGGGACTCTGGGGTTCGATAAAATAAAAGATTTTATTTAGTCTCCA3398PhePhe265GAAAAAGGGGGGAATGAAAGACCCCACCTGTAGGTTTGGCAAGCTAGCTTAAGTAACGCC3458ATTTTGCAAGGCATGGAAAAATACATAACTGAGAATAGAGAAGTTCAGATCAAGGTCAGG3518AACAGATGGAACAGCTGAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCC3578CGGCTCAGGGCCAAGAACAGATGGAACAGCTGAATATGGGCCAAACAGGATATCTGTGGT3638AAGCAGTTCCTGCCCCGGCTCAGGGCCAAGAACAGATGGTCCCCAGATGCGGTCCAGCCC3698TCAGCAGTTTCTAGAGAACCATCAGATGTTTCCAGGGTGCCCCAAGGACCTGAAATGACC3758CTGTGCCTTATTTGAACTAACCAATCAGTTCGCTTCTCGCTTCTGTTCGCGCGCTTCTGC3818TCCCCGAGCTCAATAAAAGAGCCCACAACCCCTCACTCGGGGCGCCAGTCCTCCGATTGA3878CTGAGTCGCCCGGGTACCCGTGTATCCAATAAACCCTCTTGCAGTTGCATCCGACTTGTG3938GTCTCGCTGTTCCTTGGGAGGGTCTCCTCTGAGTGATTGACTACCCGTCAGCGGGGGTCT3998TTCATTTGGGGGCTCGTCCGGGATCGGGAGACCCCTGCCCAGGGACCACCGACCCACCAC4058CGGGAGGTAAGCTGGCTGCCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACAT4118GCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCG4178TCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCGCAGCCATGACCCAGTCACGTAG4238CGATAGCGGAGTGTATACTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTG4298CACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGC4358TCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTA4418TCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAG4478AACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCG4538TTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGG4598TGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTG4658CGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGA4718AGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGC4778TCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGT4838AACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACT4898GGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGG4958CCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTT5018ACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGT5078GGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCT5138TTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTG5198GTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTT5258AAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGT5318GAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTC5378GTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCG5438CGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCC5498GAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGG5558GAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTGCA5618GGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGA5678TCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCT5738CCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTG5798CATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCA5858ACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAACA5918CGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCT5978TCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACT6038CGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAA6098ACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTC6158ATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGA6218TACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGA6278AAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGG6338CGTATCACGAGGCCCTTTCGTCTTCAA6365(2) INFORMATION FOR SEQ ID NO:2:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 264 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:MetIleGluGlnAspGlyLeuHisAlaGlySerProAlaAlaTrpVal151015GluArgLeuPheGlyTyrAspTrpAlaGlnGlnThrIleGlyCysSer202530AspAlaAlaValPheArgLeuSerAlaGlnGlyArgProValLeuPhe354045ValLysThrAspLeuSerGlyAlaLeuAsnGluLeuGlnAspGluAla505560AlaArgLeuSerTrpLeuAlaThrThrGlyValProCysAlaAlaVal65707580LeuAspValValThrGluAlaGlyArgAspTrpLeuLeuLeuGlyGlu859095ValProGlyGlnAspLeuLeuSerSerHisLeuAlaProAlaGluLys100105110ValSerIleMetAlaAspAlaMetArgArgLeuHisThrLeuAspPro115120125AlaThrCysProPheAspHisGlnAlaLysHisArgIleGluArgAla130135140ArgThrArgMetGluAlaGlyLeuValAspGlnAspAspLeuAspGlu145150155160GluHisGlnGlyLeuAlaProAlaGluLeuPheAlaArgLeuLysAla165170175ArgMetProAspGlyGluAspLeuValValThrHisGlyAspAlaCys180185190LeuProAsnIleMetValGluAsnGlyArgPheSerGlyPheIleAsp195200205CysGlyArgLeuGlyValAlaAspArgTyrGlnAspIleAlaLeuAla210215220ThrArgAspIleAlaGluGluLeuGlyGlyGluTrpAlaAspArgPhe225230235240LeuValLeuTyrGlyIleAlaAlaProAspSerGlnArgIleAlaPhe245250255TyrArgLeuLeuAspGluPhePhe260__________________________________________________________________________

	Number	Date	Country
Parent	781356	Oct 1991
Parent	720872	Jun 1991

Lymphokine gene therapy of cancer

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