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Title:
LYMPHOKINE GENE THERAPY OF CANCER
Document Type and Number:
WIPO Patent Application WO/1993/007906
Kind Code:
A1
Abstract:
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. 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 product and to secrete at least one cytokine gene product are utilized in a formulation to immunize the patient at a site other than an active tumor site.

Inventors:
SOBOL ROBERT E (US)
FRED H GAGE (US)
ROYSTON IVOR (US)
FRIEDMAN THEODORE (US)
FAKHRAI HABIB (US)
Application Number:
PCT/US1992/008999
Publication Date:
April 29, 1993
Filing Date:
October 23, 1992
Export Citation:
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Assignee:
SAN DIEGO REGIONAL CANCER CENT (US)
International Classes:
A61K38/00; A61K31/70; A61K35/12; A61K38/19; A61K38/20; A61K38/21; A61K39/00; A61K48/00; A61P35/00; C07K14/55; C12N15/19; C12N15/24; C12N15/25; C12N15/26; C12N15/85; (IPC1-7): A61K35/12; A61K39/00; A61K48/00; C12N15/19; C12N15/24; C12N15/25; C12N15/26; C12N15/63; C12N15/90
Foreign References:
Other References:
GANSBACHER B, ET AL.: "Interleukin 2 Gene Transfer into Tumor Cells Abrogates Tumorigeni city and Induces Protective Immunity", THE JOURNAL OF EXPERIMENTAL MEDICINE, ROCKEFELLER UNIVERSITY PRESS, US, vol. 172, 1 October 1990 (1990-10-01), US, pages 1217 - 1224, XP002982887, ISSN: 0022-1007, DOI: 10.1084/jem.172.4.1217
TEPPER R I, PATTENGALE P K, LEDER P: "MURINE INTERLEUKIN-4 DISPLAYS POTENT ANTI-TUMOR ACTIVITY IN VIVO", CELL, CELL PRESS, US, vol. 57, 5 May 1989 (1989-05-05), US, pages 503 - 512, XP002940796, ISSN: 0092-8674, DOI: 10.1016/0092-8674(89)90925-2
FEARON E R, ET AL.: "INTERLEUKIN-2 PRODUCTION BY TUMOR CELLS BYPASSES T HELPER FUNCTION IN THE GENERATION OF AN ANTITUMOR RESPONSE", CELL, CELL PRESS, US, vol. 60, no. 03, 9 February 1990 (1990-02-09), US, pages 397 - 403, XP001108962, ISSN: 0092-8674, DOI: 10.1016/0092-8674(90)90591-2
OGURA H., ET AL.: "IMPLANTATION OF GENETICALLY MANIPULATED FIBROBLASTS INTO MICE AS ANTITUMOR ALPHA-INTERFERON THERAPY.", CANCER RESEARCH, AMERICAN ASSOCIATION FOR CANCER RESEARCH, US, vol. 50., no. 13., 1 January 1990 (1990-01-01), US, pages 5102 - 5106, XP000906780, ISSN: 0008-5472
GANSBACHER B., ET AL.: "RETROVIRAL VECTOR-MEDIATED GAMMA-INTERFERON GENE TRANSFER INTO TUMOR CELLS GENERATES POTENT AND LONG LASTING ANTITUMOR IMMUNITY.", CANCER RESEARCH, AMERICAN ASSOCIATION FOR CANCER RESEARCH, US, vol. 50., no. 24., 15 December 1990 (1990-12-15), US, pages 7820 - 7825., XP000197170, ISSN: 0008-5472
See also references of EP 0668781A4
Attorney, Agent or Firm:
Campbell, Cathryn (Suite 700 4370 La Jolla Village Driv, San Diego CA, US)
Download PDF:
Claims:
89 WE CLAIM:
1. A method of treating cancer in a patient comprising the stimulation of that patient's immune response against the cancer by immunizing said patient at a site other than an active tumor site with a formulation comprising tumor antigens and CE cells genetically modified to express at least one cytokine gene product.
2. The method of claim 1 wherein tumor cells previously isolated from said patient provide the tumor antigens.
3. The method of claim 1 wherein the cytokine gene is selected from the group consisting of interleukin 1, interleukin2, interleukin3, interleukin4, interleukin5, interleukin6, and gammainterferon.
4. The method of claim 3 wherein one cytokine gene is interleukin2.
5. The method of claim 1 wherein at least one cytokine gene is transferred into cells to generate CE cells by recombinant methods.
6. The method of claim 5 wherein the cytokine gene is present in an expression vector.
7. The method of claim 6 wherein said expression vector additional contains a suicide gene.
8. The method of claim 5 wherein the CE cells are generated from fibroblasts and antigenpresenting cells. 90 .
9. A method for enhancing a patient's immune response to a cancer comprising: a) isolating fibroblasts from said patient; b) culturing said fibroblasts in vitro; c) transducing said fibroblasts with a retroviral expression vector containing the gene coding for IL2 and a gene coding for a tumor antigen in a retroviral expression vector, to express said tumor antigen and to express and secrete said IL2 by said fibroblasts; and d) immunizing said patient with said fibroblasts that express IL2 at a level sufficient to enhance an immune response but low enough to avoid substantial systemic toxicity and that express said tumor antigen, at a site other than an active tumor site.
10. The method of claim 9 wherein said fibroblasts are further modified to express a suicide gene.
11. A composition for increasing a patient's immune response to tumor antigens comprising tumor antigens and CE cells genetically modified to express at least one cytokine gene product.
12. The composition of claim 11 wherein the cytokine gene is selected from the group consisting of interleukin1, interleukin2, interleukin3, interleukin4, interleukin5, interleukin6, and gamma interferon.
13. The composition of claim 12 wherein one cytokine gene is interleukin2.
14. The composition of claim 11 wherein each cytokine gene is expressed at a level sufficient to stimulate the immune response but low enough to avoid substantial systemic toxicities.
15. The method of claim 9 wherein in said transducing step said retroviral expression vector has a promotor causing sustained secretion of IL2.
16. The method of claim 15 wherein said retroviral expression vector causes the secretion of at least four units of IL2 per day for a period of ten days or longer.
Description:
Lymphokine Gene Therapy of Cancer

BACKGROUND

This application is a continuation-in-part of United States Patent Application Serial No. 07/781,356, filed on October 25, 1991, which is a continuation-in-part of United States Patent Application Serial No. 07/720,872, filed on June 25, 1991, both of which are incorporated herein in their entirety.

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 ade a) limit the dose and efficacy of intravenous and intralymphatic IL-

- TE SHEET

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 (α-INF) and gamma interferon (γ-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 tu origenicity of the cytokine-secreting tumor cells when implanted into syngeneic hosts. The transfer of genes for IL-2 (11,12) γ-INF (13) or interleukin-4 (IL-4) (14) significantly reduced or eliminated the growth of several different histological types of urine 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

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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 subσutaneously, 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

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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

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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

Figure 1 shows schematic diagrams of retroviral vectors DC/TKIL2, LXSN-IL2, and LNCX-IL2.

Figure 2 shows a mean IL-2 concentration of triplicate supernatant samples measured by ELISA. Supernatants were harvested from overnight cultures of approximately 1.5 x 10 6 semi-confluent fibroblasts.

SUBSTITUTE SHEET

Figure 3 shows biological activity of the IL-2 secreted by the transduced fibroblasts was demonstrated by measuring mean 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 x 10 6 semi-confluent fibroblasts.

Figure 4 shows comparisons between animals injected with 10 5 CT26 tumor cells alone (D) , 10 s CT26 tumor cells and 2 x 10 6 unmodified BALB/C fibroblasts (I) ; 10 5 CT26 tumor cells and 2 x 10 6 IL-2 transduced BALB/C fibroblasts (§); and 10 5 CT26 tumor cells and 1 x 10 6 transduced BALB/C fibroblasts (o) . 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.

Figure 5 shows PCR analysis of neomycin phospho- transferase 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) .

Figure 6 shows the effect of IL-2 modified fibroblasts on tumor establishment and development using 2 x 10 6 fibroblasts mixed with 5 x 10" CT26 tumor cells concentrating on the rate of tumor growth.

Figure 7 shows the effect of IL-2 modified fibroblasts on tumor establishment and development using 2 x 10 6 fibroblasts mixed with 5 x 10" CT26 tumor cells concentrating on the time of tumor onset for the individual animal in each treatment group.

p-UDC STiTUTE SHEET

Figure 8 shows the effect of IL-2 modified fibroblasts on tumor establishment and development using 2 x 10 6 fibroblasts mixed with 1 x 10 5 CT26 tumor cells concentrating on the rate of tumor growth.

Figure 9 shows the effect of IL-2 modified fibroblasts on tumor establishment and development using 2 x 10 6 fibroblasts mixed with 1 x 10 5 CT26 tumor cells concentrating on the time of tumor onset for the individual animal in each treatment group.

Figure 10 shows the effect of IL-2 modified cells on tumor establishment and development using 2 x 10 s DCTK- IL2-modified CT26 tumor cells mixed with 1 x 10 5 unmodified CT26 compared to 2 x 10 6 DCTK-IL2-modified fibroblasts mixed with 1 x 10 5 CT26 concentrating on the rate of tumor growth.

Figure 11 shows the effect of IL-2 modified cells on tumor establishment and development using 2 x 10 s DCTK- IL2-modified CT26 tumor cells mixed with 1 x 10 5 unmodified CT26 compared to 2 x 10 6 DCTK-IL2-modified fibroblasts mixed with 1 x 10 5 CT26 concentrating on the time of tumor onset for the individual animal in each treatment group.

Figure 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 x 10 6 fibroblasts mixed with 2.5 x 10 s irradiated CT26 tumor cells 7 days prior to challenge with 5 x 10 4 fresh tumor cells.

Figure 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 x 10 6 fibroblasts mixed with 2.5 x 10 5 irradiated CT26 tumor cells 7 days prior to challenge with 5 x 10 4 fresh tumor cells.

SUBSTITUTE SHEET

Figure 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 x 10 6 fibroblasts mixed with 2.5 x 10 5 irradiated CT26 tumor cells 14 days prior to challenge with 5 x 10 4 fresh tumor cells.

Figure 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 x 10 6 fibroblasts mixed with 2.5 x 10 5 irradiated CT26 tumor cells 14 days prior to challenge with 5 x 10 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 iinmune 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.

SUBSTITUTE SHEt

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.

SUBSTITUTE SHEET

(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, γ-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 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

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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 R ) gene. Following

SUBSTITUTE SHEET

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 urine 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 Immunolo ical 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 ho ogenates 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.

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(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 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 7 intact tumor cells. The tumor preparation should be mixed with numbers of CE or carrier

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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.

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EXAMPLE I

IMMUNIZATION WITH FIBROBLASTS EXPRESSING IL-2 MIXED WITH IRRADIATED TUMOR CELLS

1) Isolation of Autolocrous 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) Retroviral 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 R ) genes. An N2 vector containing the Neo 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

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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 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°C until required for subsequent use in immunizations.

Table 1 Adventitious A ents and Safet Testin

SUBSTITUTE SH«=T

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 50 , C 3 and C 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 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.

SUBSTITUTE SHEET

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 7 tumor cells will be mixed with 10 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 mannert

SUBSTITUTESHEET

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.

Schematic diagrams of the structure of these retroviral vectors are provided in Figure 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 and 4.

SUBSTITUTESHEET

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) . Figure 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 β-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 3 H-thymidine was measured as an indicator of cell proliferation and IL-2 activity (Figure 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

SUBSTITUTESHEET

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 (Figure 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 Figure 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 x 10 6 transduced fibroblasts and CT26 tumor cells (Figure 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 Figure 1). The normal human IL-2 leader

SUBSTITU

sequence has been replaced with a chimeric sequence containing rat insulin and human IL-2 leader sequences (see Tables 2, 3 and 4) . This chimeric leader sequence enhances IL-2 gene expression.

To construct the LXSN-RI-IL2 vector, the bacterial plasmid pBCl2/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 Hindlll 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 Nal at 55°. After cooling to room temperature, 4 μl of oxidized silica solution (BIO-101, La Jolla, CA) was added to adsorb the DNA. The silica was ythen washed with a cold solution of 50% ethanol containing 0.1 M NaCl in TE buffer. The DNA was eluted from the silica by heating at 55° in distilled H 2 0. The purified IL- 2 cDNA was then directionally ligated into the Hpal-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.

SUBSTITUTESHEET

Table 2

Description of (he LXSN-RI-IL2 from position 1 to 6365

a

SUBSTITUTE SHEET

Table 3

Enzyme [# Cuts] Position(s)

2] 1961, 2481

2] 811, 6295

1] 4252

191 392, 394, 445, 969, 971, 1193, 3052, 3084, 3807, 3809, 4081, 4083, 4527, 5108, 5438, 5931, 6263

5] 808, 2685, 3860, 5910, 6292

13] 260, 273, 328, 626, 756, 1277, 3676, 3689, 3744, 4041, 5511, 5733

4] 34, 1064, 1955, 3446 2] 1592, 4480

20] 161, 237, 473, 474, 602, 644

2689, 2849, 3578, 3653, 3888, 3889,

4059, 4126, 4161, 4860, 5556, 5907

5] 808, 2685, 3860, 5910, 6292

3] 5239, 5258, 5950

33] 29, 33, 119, 190, 411, 654,

742, 1470, I486, 1751, 1935, 2003, 2446,

2791, 3249, 3441, 3445, 3532, 3607,

4069, 4122, 4141, 4422, 4648, 4738,

5041, 5562, 5662, 5725

20] 1110, 1414, 1665, 2018, 2147, 2160, 2553, 2864, 2929, 3110, 4027, 5041, 5129, 5225, 5226, 5689, 6006, 6010

4] 231, 3572, 3647, 4896

2] 847, 1076

19] 323, 413, 426, 597, 1583, 1721, 2724, 2798, 2988, 3050, 3739, 3828, 4012, 4300, 4798, 5959, 6044

2] 2787, 5595 4] 1717 , 4296, 4794 , 6040

SUBSTITUTE SHEET

Apyl [ 22] 315, 623, 801, 814, 1227, 1252, 1275, 1295, 1325, 1526, 1536, 1558, 1630, 2196, 2251, 2268, 3072, 3731, 4038, 4508, 4629, 4642

Aqul [ 6] 241, 472, 1998, 3821, 3854, 3887

Asel t 2] 1801, 5545

Asp700 [ 1] 5972

Asp718 [ 2] 476, 3891

AspAl [ 1] 1145

Asul [ 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, 6349

Aval [ 6] 241, 472, 1998, 3821, 3854, 3887 Ava2 [ 13] 260, 273, 328, 626, 756, 1277, 3201, 3676, 3689, 3744, 4041, 5511, 5733

Ava3 [ 2] 2232, 2304

Avr2 [ 2] 1962, 2482

Ball [ 3] 658, 1169, 2767

BamHl t 1] 2152

Banl t 9] 318, 476, 1200, 2684, 2719, 3734, 3859, 3891, 5321

Ban2 [ 8] 413, 426, 597, 1583, 3050, 3828, 3841, 4012

Bbel [ 2] 2688, 3863 Bbvl [ 22] 969, 997, 1738, 2493, 2632, 2758, 2800, 2816, 2909, 3321, 4060, 4131, 4228,

4372, 4390, 4809, 4899, 4902, 5108, 5411,

5600, 5802

Bell t 1] 2526 Bgll t 2] 2435, 5493

Bspl286I [ 19] 323, 413, 426, 597, 1583, 1721,

2631, 2724, 2798, 2988, 3050, 3739, 3828, 3841, 4012, 4300, 4798, 5959, 6044

SUBSTITUTE SHEET

BspHl [ 3] 5200, 6208, 6313 BspMl C 4] 1501, 2500, 2572, 2953 BssH2 [ 4] 392, 443, 3082, 3807 BstE2 [ 1] 1145 BstNl [ 22] 315, 623, 801, 814, 1227, 1252, 1275, 1295, 1325, 1526, 1536, 1558, 1630, 2196, 2251, 2268, 3072, 3731, 4038, 4508, 4629, 4642

BstUl [ 19] 392, 394, 445, 969, 971, 1193, 2751, 3052, 3084, 3807, 3809, 4081, 4083,

4186, 4527, 5108, 5438, 5931, 6263

BstXl [ 1] 2060 BstYl [ 11] 2010, 2152, 2521, 2856, 3102, 5121, 5132, 5218, 5230, 5998, 6015

Bsu36I 2] 847, 1076

Ccrl 1] 1998

Cfol [ 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, 6265

Cfrl [ 9] 656, 790, 1167, 1188, 2591, 2765, 3156, 3183, 5761

CfrlOI [ 3] 3004, 3185, 5453 Cfrl3l [ 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, 6349

Cvnl [ 2] 847, 1076 Ddel [ 23] 75, 165, 191, 282, 553, 847,

1076, 1348, 1692, 2442, 3348, 3487, 3582,

3657, 3698, 3879, 3967, 4290, 4755, 5164,

5330, 5870, 6296

Dpnl [ 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, 6053

SUBSTITUTESHEET

Dral [ 3] 5239, 5258, 5950 Dra2 t 4] 328, 1277, 3744, 6349 Eael [ 9] 656, 790, 1167, 1188, 2591, 2765, 3156, 3183, 5761

Eagl [ 2] 790, 2591 Eco47l [ 13] 260, 273, 328, 626, 756, 1277, 3201, 3676, 3689, 3744, 4041, 5511, 5733

Eco52l [ 2] 790, 2591

Eco81I [ 2] 847, 1076

EcoNl [ 2] 850, 1450

Eco0109l [ 4] 328, 1277, 3744, 6349

EcoRI [ 1] 1460

EcoRI* [ 14] 938, 1037, 1460, 1798, 1805, 1928,

2064, 2121, 2236, 2308, 2400, 5240, 5546, 5801

EcoR2 [ 22] 313, 621, 799, 812, 1225, 1250,

1273, 1293, 1323, 1524, 1534, 1556, 1628, 2194, 2249, 2266, 3070, 3729, 4036, 4506, 4627, 4640

EcoR5 [ 4] 137, 213, 3554, 3629 ECOT22I [ 2] 2232, 2304 Fdi2 [ 2] 2787, 5595 Fnu4Hl [ 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, 6115

FnuD2 [ 19] 392, 394, 445, 969, 971, 1193, 2751, 3052, 3084, 3807, 3809, 4081, 4083, 4186, 4527, 5108, 5438, 5931, 6263

Fokl [ 13] 498, 1198, 1358, 1679, 2333, 2552, 3009, 3034, 3912, 4168, 5339, 5520, 5807

Fspl [ 2] 2787, 5595 Hae2 [ 4 ] 2688 , 3863 , 4358 , 4728

SUBSTITUTE SHEET

Hae3 [ 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, 6350

Hap2 [ 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, 5907

Hgal [ 8] 455, 707, 960, 1580, 4175, 4591, 5169, 5899

HgiAl [ 9] 413, 1721, 2798, 2988, 3828, 4300,

4798, 5959, 6044

Hhal [ 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, 6265

HinPl t 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, 6263

Hinc2 1] 5914 Hind2 1] 5914 Hind3 1] 2498 Hinfl [ 14] 298, 517, 857, 868, 1553, 1814,

3170, 3304, 3356, 3881, 4380, 4455, 4851, 5368

Hpa2 [ 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, 5907

Hphl [ 11] 1214, 1240, 1817, 2863, 4102, 4111,

5216, 5443, 5859, 6065, 6100

Kpnl C 2] 480, 3895 Mael [ 15] 30, 293, 689, 727, 739, 1452, 1606, 1893, 1963, 2483, 3442, 3709, 4975, 5228, 5563

HEET

Ndel [ 1] 4303 Nde2 [ 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, 6051

Nhel [ 3] 29, 1605, 3441 Nla3 [ 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, 6317

Nla4 [ 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, 6259

Nsil i 2] 2232, 2304

Nsp(7524)l[ 8] 1596, 1835, 1856, 2230, 2302, 3090,

4119, 4484

Nsp(7524)2[ 19] 323, 413, 426, 597, 1583, 1721,

2631, 2724, 2798, 2988, 3050, 3739, 3828, 3841, 4012, 4300, 4798, 5959, 6044

NspB2 [ 12] 119, 190, 1751, 2158, 2791, 3532, 3607, 3989, 4192, 4822, 5067, 6008

NspHl [ 8] 1596, 1835, 1856, 2230, 2302, 3090, 4119, 4484

PaeR7I [ 1] 1998 Pall [ 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, 6350

Plel [ 7] 865, 1547, 3350, 3889, 4374, 4859, 5362

PpuMl [ 3] 328, 1277, 3744 Pssl [ 4] 331, 1280, 3747, 6352 PstI [ 6] 987, 1163, 1888, 2511, 2738, 5618 Pvul [ 1] 5743

HEET

in co to en cn co tO «J r-T VO 4* -> to

4* t tn co to co co vo cn>—'

NJ CO 4* O 4* CO t to co CO * 4* to w CO to co cn 4* to in tn cn 4* in O M CO 00

C en ** vo- H vo cn co to CO c to 00 in

H to CO 4* t to o o to vo o πi co oo-

M 4

ΓΛ to rπ co VO o 4* CO VO m t CO O -4

H r-i ιt»- -400 co o cn vo 43. to in o CO M -O in M- -J 4*

— — to

M 4* CO O in to -i

-4 tO- -4 CO to to

CO vo

Sspl [ 1] 6177 Sstl [ 2] 413, 3828 Stul [ 2] 1961, 2481 Styl [ 9] 324, 536, 1303, 1962, 2389, 2482,

3117, 3740, 3950

Taql [ 15] 860, 1096, 1407, 1418, 1660, 1999,

2514, 2798, 2954, 2978, 3014, 3176, 3367,

4580, 6024

Thai [ 19] 392, 394, 445, 969, 971, 1193,

2751, 3052, 3084, 3807, 3809, 4081, 4083,

4186, 4527, 5108, 5438, 5931, 6263

Tthllll [ 6] 465, 877, 1275, 2803, 3880, 4227 Xbal [ 2] 1892, 3708 Xhol [ 1] 1998 Xho2 [ 11] 2010, 2152, 2521, 2856, 3102, 5121,

5132, 5218, 5230, 5998, 6015

Xmal [ 2] 472, 3887 Xma3 [ 2] 790, 2591 Xmnl [ 1] 5972 Xor2 [ 1] 5743

SUBSTITUTE SHEET

Table 4

Enzymes which do not cut LXSNRII.L2:

Acc3 Bgl2 Clal Hpal Nrul SnaBl

Apal Bsml Dra3 Mlul PflMl Spll

Asu2 BspM2 Eco47III Mrol Sac2 Sst2

Ban3 BstBl Espl Notl Sail

SUBSTITUTE SHEET

i

C rH

A a

EH

I TUTE SHEET

Asul —33 112-+1-11—11—+ -4—1—-241—+1 + 12—1—+

Aval -+ 21+ 4- 4.

Ava2 ___3 11 __ .___ 1 + -21—+1- -+-

Ava3 11- -4--

Avr2

Ball

BamHl 4- -_l 4. +-

Banl ■1-1 +—1 + 11—+ 111+-

Ban2 -11-1—+ 1 + +1 2-1-

Bbel 1-+-

Bbvl 2 1—+ 1-1121+ 1 +111-2 12+1 1-1—1-+ + + 1 + + + + 6I 111-1—+ 1-1—+ 111-11 12-1—1 1-+—- 11 +-1 + 1 4- 11 1 11 + + +1

-+-1- 1 χ-2-—23—31 +—3 +1 1—+1 1-2 + + 21 2—1 + 1—+2 2-+21 1 +1 1 1+-

22—1—1 + 211-+3—1 3-+11111—111-11 11 1+

CfrlOI

Cfrl3l —33 112-+1-11— 11 + +— 1 241— +1 + 12 — 1— +

Cvnl 1+1 + + + + +

Ddel 1111 — 1 — 1+1— 1—1— + 1 + 1-1111-11—1 1— +-1-1 1-+

Eagl •1-+-

Eco47I

Eco52l 1-4- 4. 1 4. 4. 4. 4.

M

ON M

H O * CN rH rH rH rH rH rH CN m CN B3 CN rH rH CN CN CO rH

CO iS O K K PJ K E N^ αriWCNncSH rtlH ft ϋ'OTJWNrtHHtNi nHCVlHHHrtC

0 0 0 0 0 0 0 0 -rl 3 :_I^ ftCD CD fttrj-H ( u , 3 β 3 β 3 ( u'-S S Cϋ CD CD 0 0rH tD ft-μ +J 0 ϋ ϋ 0 ϋ ϋ ϋ ϋ T3 β B 0 ω trJ ( β td σι CnΛ -H -H -H -H -H ft ft ft (d <d (OA A 3 CO CO CO CD wpqwMWHHwfafafafafaffiKE-π-πKffiK-πmm

SUBSTITUTE SHEET

rH H rH rH rH rH rH rH CN rH CO f rH H l-t H rt g H H H Id CD ) H-H O CD CD CD td ld-r| (d rl 3 O (d-'-< ld CU cd td o uO lx:HH to u o'TJ CD l H>H e ft

S2SSB2l3|32gg -( ri i i W W WWWWWWWW

SUBSTITUTE SHEET

1-HrHrHrHrHι-HrHrHrHrHCNrHC rHCN

Λ ft-P 3 ιCT 1 ( d.3 ( d 0 0 <d <d 3 ftto ω-μ-μ rfΛ+JΛΛΛ ε ε E O C Q C Q Q Q EH EH OOϋXXXtX

SUBSTITUTE SHEET

41

42

Table 5 (Cont ' d)

fro* 1 to C34S. Nu«bara4 froa poaltlon 1.

UtSNKXl -10004- -30004' •30004- -40O04- -S0004 -KutV f • long tar

I«Pllt| I to 443 of AXXtt naoaryal* phoβpKotr* -MuXΛT J* long tor olαnal 1 1 9atX2 4_1

■•mi 1 1-2-4—23—31 ■•tυi 21 2—1 21—1 ■■txi ...... — ...«.-.. — ......

Bβtϊl ...... — .- - — ........ .1—1.

Bau36X 141 — — — —

Ccrl ...........4- — — - — — .....

Cfol 22—1—1 - 211- 3—1 3- 11111 — 111-

Cfrl - 1-1-4-11 1-1— -2

CfrlOX --— — -— — ............. -1

Cfrl3X —33 112-41-11—11 — 1 241— 1

Cvnl 141 Ddal 1111—1 141—1 1— 1-1111-1 •1— Dpnl -1 4-11-1—1 -1 3 2 2 1 — Oral — — — 4— — — Dra2 1 X • -.••I 1-1-4-11 .1-1. - Baαl 1-4 -1 — Cco4?I 3 11—4 1 •21- - tcosax 1-4 -

Ecoβll 141

CcoNl 14 1 -

BCO 109X J 4 1 -

EcoAl 4 J -

Icoftl* 11 1 1 mil- • 4

*CO*2 1 — i-j.4-.22— 31— — j — 1. 1-2- •

BcoftS -11 4 ——11— -

Table 5 (Cont ' d) fro* 1 to 434S. ITu-vbara froa poit to* 1.

LXSMIIXXU -10004. -30004- -30004- -40004- -S0004- •40004— -. -KutV f IOAO tar -> I«X»1U| I to 443 Of AXXL3 nooajyolo phoaphotra -Mal T 2' l ng tar •I na

■i-fi J .4—

■lafl — 1 .1 J 1—1-4- ——4-1-2 1 11 1-4 1 —-*-.

•1 1—1-

Kao3 — -421-1-1— 1—4- 1-4.1 — *-ll 111 — 1- -2-1-1

Kbol -1 »-u-ι— 1 — 1-1— —3— 1121— — 1 — 1 _-»ι 3 2i. -21—21

K o2 1 4-1—1-11—24 41-11— 2-1-1

Knl 1-131—11—117211—24 242-1— 41— — 113121—1-11—14 111—1—4 1

Meal 1 41-1 2-2-2- 4—1— —4—1— ——4-lJl— 2 14 — -1

■■pi -11—1-2-1-4 4 112111-1 — -11—1112 1-2-41 1111—14

Tab le 5 (Cont ' d )

free 1 to 4341. Rumbarad froa poaltto A 1.

U HR1IL2 » 1000«- -2O004. -30004. -40004. -S0004- -40004 -MuCV § lonα tar *>

C«pUt]

I to 443 of XX XU nooayeia phoaphotra -KutV J' long tor olαnal

Matl 4 4 1-4 4

Mat! 14 4 -4 4

Kval 1 1-2-4—23—31 4—3 -41 1—41 1-2

Haal 4 4-1 4

Karl 4 1 4 '-1-4

Hel l -1 .-2.3-1. 4 ll-4 11—2112 1-

Mcol —4 4 .1— 4-1— 4

Ndal 4— ♦ 1

Mda2 -1 4-11-1—1—1-1 3—1121 1 1 - 2221—21 — 1—

Mhal 1 ♦ 1 4 1 ♦

Nla3 1 4—1—11-1141-111-1 1«12--- 1 4-H— 1 1 211- -1-1

Mla4 -112-1-1-2-1—11 4-111 U— 4 111141 U —12—1 — 1-

Nail 4 - — 11 4 4

Map( 7524 ) 1 1—114 — H 1 4-1 1

Mep(?S24 )2 111-1 4 1-1—4 111-11 12-1 1 1 •11-

XapB2 -11- 1—4-1 .1.4 2 1-1 -1-41 — 1-

XapHl •1—114—11 H -1 1.

»*aft?X 1 ...4— — — 4 — —

Pall —j 1-3-4112 11—11 221-1—4-2 22 4 3 -11— 1- 1

»lal 4 1 4 4 1 14 1 1 •1

PpuMl 1 4 1 4 4 1—4

Peal 1 4 1 4 4 1—4 1

Pvu2 .11 1—4 1-4 —2—4

Table 5 (Cont 'd) froa 1 to 434S. ffuaJ ara4 froa poaltton 1.

U-SNRXIL2 -10004- -20004- -30004- -40004- -50004- -40004 -NuffV f long tar -> Ilpl l t to 402 f A1XL3 noooyolx phoaphotra -KttLV 2' long tar •lgnal 1 Raal •11—1- .4... 11- — 1— -14 - .4 -4 X4ar2 — .... -.— .4— — — . ...4. .4—— — — — Saul ..-4. .4— — — 4— — SatiSAl -1 —4-11-1—1 •1121- — 1. -42221 21—21— Saut4X —33 112-41-11—11 ...........4—1—241—41 — ——-4—12—1—4- — 1 Seal — — .....4— ..... — — .——4— ——4- ——4 —1-4 — Scrri -111-2-3-3-4—23—31— —3 11-41 -111-2122—1-2-1-4 1 14 S ul 111-1 1-1— 111-11 12-1 — 1 1-4 11 4--- Sael -112-112-5-4— 14— 2 —2-31 1-4-1 112-112 1—4 4 SfaJU —1—1 1 1— -111 1—1111-1 1—14-112—1 - 1-1-41 Sfl —————4—...... ....1— ....4.— .... 4 — ——4 — 4 Saval

•tal

fro* 1 to 4345. Xunvbarad froa position 1.

>Khal >Afl3 tout* OKA tnd/KO-KuSV . DMA. start. (Split) j

I 10 20 30 40 SO to 70 y * • • • • y* y* • • • • • • •

TTTCAAACAC CCCACCOCTA GCTCCCAACC TACCTTAACT AACCCCACTT TCCAACCCAT CCAAAAATAC AAACTTTCTC CCCTCCCCAT CCACCCTTCC ATCGAATTCA TTCCCCTCAA ACCTTCCCTA CCTTTTTATO

>PYU2

>N«pB2 >EcoRS

•0 90 100 110 120 130 (l40

ATAACTCACA ATACCAAACT TCACATCAAC CTCACCAACA AACAAACACC TCAATACCAA ACACCATATC TATTCACTCT TATC TTTCA ACTCTACTTC CACTCCTTCT 1TCTTTCTCC ACTTATCCTT T TCCTATAO

>PVΛI2 SO 140 170 110 ItO 200 210

TCTCCTAΛCC CCTTCCTCCC CCCCCTCACC OCCAACAACA CATCACλCAC CTCACTCATC CCCCAAACAO ACACCATTCC CCAACCλCCC CCCCCACTCC CCCTTCTTCT CTACTCT6TC CACTCACTAC CCCCTTTCTC

>λval

XcoftS >Alw«l >λq l j . 220 230 I 340 | 350 240 270 210

CATATCTCTC CTAACCACTT CCTCCCCCCC CTOCCCCCCA ACAACACATC CTCCCCACAT COCCTCCAOC CTATACACAC CATTCCTCAλ CCACCCCCCC CACCCCCCCT TCTTCTCTAC CAGOOCTCTA CCCCACCTCO

>rp xι

>0ra2

XeoOlOfX

>Baal >Styl >Pι)il >Rβal

290 300 310 320 | 330 | 340 |3S0

CCTCACCACT rXCXACTCAA TCATCACATO rTTCCACOCT OCCOCA OCA OCTCAAAATΘ ACCCTCTACC OCACTOCTCA AACATCACTT ACTACTCTAC AλλOCTCCCλ CCCCCTTCCT CCACTTTTAC TCCCACATOO

Table 6 (Cont ' d)

>Ban2

*

>Sael

*

>B«βH2 >RgiAl

360 370 340 390 j 400 410 | 420

• • • • • • • • y • • • • y • . •

TTATTTCAAC TAACCAATCA CTTCCCTTCT CCCTTCTCTT CGCWCXrCTTC CCCTCTCCCA CCTCAATAAA AATAAACTTC ATTCCTTACT CAACCCAACA CCCAACλCAA CCCCCCCAAC CCCAGACCCT CCAGTTATTT

>Alp714

>B*nl

ACACCCCACA ACCCCTCACT CCCCCCCCCA CTCTTCCCAT ACACTCCCTC CCCCCCCTAC CCCTATTCCC TCTCOCCTCT TCCCCACTCA CCCCCCCCCT CACAACCCTA TCTCACCCAC CCCCCCCATC CCCATAAGCC

>Styl 500 S10 520 530 | 540 SSO 560

• • • • 4 • • »ψ • 4 • • •

AATAAACCCT Cl tf«Uwi rt CCATCCCAAT CCTCCTCTCO CTCΪTCCT O CCACCCTCTC CTCTCACTCA TTATTTCCCA CAACCACAAA CCTACCCTTA CCACCACACC CACAAGCAAC CCTCCCλCAO CACACTCACT

>8an2

570 580 590 |C00 €10 620 €30

TTCACTACCC ACGACCCCGO TCTTTCATTT CCCCCCTCCT CCCCCATTTC CACACCCCTC CCCACCCACC AACTCATCCC TCCTCCCCCC ACAAACTAAA CCCCCCλCCλ CCCCCTAAAC CTCTCGCCλC CCCTCCCTCO

>B*11

ACCCACCCAC CACCCGCACC TAACCTCCCC ACCAACTTAT CTCTCTCTCT CCCATTCTCT ACTCTCTATC TCCCTCCCTG CTCCCCCTCC ATTCCACCCC TCCTTCAATA CACACACACA CCCTAACACA TCACACATAC

>Spal

>Hgal >R*al

*

I 710 720 | | 730 740 750 740 770

TTTCATCTTA TCCCCCTCCC TCTCTACTλO TTλCCTλλCT ACCTCTCTAT CTCCCCCACC OCTCCTCCλA AAACTACAAT ACCCCCACCC ACλCATCλTC AATCCATTCA TCCACACATA CACCCCCTCC CCλCCACCTT

>teoS21

CTOACCACTT CTCλλCλCCC OCOCCCλλCC CTCCCACλOO TCCCACCCλC TTTCCCCCCC CTTTTTCTCO CACTOCTCAA CACTTCTCCO CCCCCCTTCO OACCCTCTOC λCCOTCCCTO AAλCCCCCOO CλλλλλCλOC

>BC0N1

CCCCACCTCA CCAACCCACT CCATCTCCAA TCCCACCCCC TCACCATATC TCCTTCTCCT ACCAGACCAC CCCCTCCACT CCTTCCCTCA CCTACACCTT ACCCTCCCCC ACTCCTATAC ACCAACACCA TCCTCTCCTC

>Hg«l

920 930 940 9S0 960 970 940

AACCTAAAAC ACTTCCCCCC TCOCTCTCAA l U _ .t ... I CCCTTTCCAA CCCAACCCCC CCCTCTTCTC TTCCATTTTC TCAACCCCCC ACCCACACTT AAλλACCλλλ CCCAAACCTT CCCTTCCCCC CCCACAACAC

>P*tl o

1990 1000 1010 1020 1030 1040 10S0

TCCTCCACCA TCCTTCTCTC TTCTCTCTCT CTCACTCTCT TTCTCTATTT CTCTCλAAAT TACCCCCACA ACCACCTCCT ACCλλCACAC AλCACλCλCλ CλCTCλCACA AACACATAAA CACλCTTTTλ ATCCCCCTCT

>λocl

*

>Sιul

Λ

>CTBl

*

>8tu3€X >Afl3 >Ieo41X lO€O I 1070 1 080 1090 1100 1110 1120

CTCTTACCAC TCCCTTλλOr TTCACCTTAO CTCλCTCCλλ λCATGTCCλO CCCATCCCTC λCλλCCACTC CACAATCCTO λCCCλλTTCA λλCTCCλλTC CACTCACCTT TCTλCλCCTC CCCTACCCAO TCTTCCTCλO

>Cfrl >λ«pλl >Eaal >E*tl

Table 6 (Cont ' d )

>K-iβ2 >BatE2 >Pβtl |>Ball >Ka β 2 > frl

1130 1140 I 1150 1160 | 1170 1140 1190

4 4 • y« * • t • y • y y« β 4 • y •

CCTλCATCTC AACAACACAC CTTCCCTTAC CTTCTϋCTCT CCACAATCCC CAACCTTTAA CCTCCCATCG CCATCTACAC TTCTTCTCTC CAACCCAATC CAACAOCACA OCTCTTACCC CTTCCAAATT CCACCCTACC

>Rphl

1230 1240 1250 1260

COCCCACAOC CCACCTTTλλ CCCACACCTC ATCACCCACC TTAACATCAA CCTCTTTTCA CCTCCCCOCC t -OCCTCTCC CCTCCAAATT CCCTCTCCAO TAGTCCCTCC AATTCTACTT CCAGAAAACT CCλCCCCCCC

1270 1310 1330 1330

ATGCACACCC ACACCACCTC CCCTACATCC TCACCTCCCA ACCCTTCCCT TTTCACCCCC CTCCCTCCCT TλCCTGTGOC TCTCCTCCAC GCCATCTACC ACTCCλCCCT TCCCAACCCA AAACTCCCOC CACCCACCCA

o

1340 I 1350 1360 1370 1380 1390 1400

CAACCCCTTT CTACACCCTλ ACCCTCCCCC TCCTCT ICCT CCATCCCCCC CCTCTCTCCC CCTTCAACCT CTTCGCCAAA CATCTCCCAT TCCCACCCCC λCCACAACCλ CCTACCOCCC CCACAGACCC CCAACTTCCλ

>Eco*l >feoRl

1410 1420 1430 1440 14S0 1440 1470

CCTCOTTCCλ CCCCCCCTOO ATCCTCCCTT TATCCACCCC TCACTCCTTC TCTAOOOCOO AATTCCTTAC CCACCAACCT CCCCCCCAGC TAOCλCCCλλ ATACGTCCCC ACTCACCAAO λCATCCOCCC TTAACCAATC

Table 6 (Cont ' d)

>B«pMl >*«»1

1480 1490 1S00 | 1510 1520 1530 1540

CTTGCTAACT CλCCλCCTAC ACTOCCAAAC CATCACCAAO CλCCTATCTλ CTCTCCACCG TCCCCCTCCC CAACCATTCΛ CTCGTCCATC TCACCCTTTC CTACTCCTTC CTCCATACAT CACACCTCCC ACCCCCACOG

>Rsal

610

TTCCCCACTC AACACTCCAC CCATTTCλCC CACCCTCTCG ©CTCTTCTCT TACATCTACC TTTTCCTACC AACCCCTCAC TTCTCACCTC CCTAAACTCC CTCCCACACC CCACλλCλCλ ATCTACATCG λλλλCCATCβ

1620 1630 1640 1650 1660 1670 1680

CTCAACCCTC λCTλTCTTCC λCCTCATTCT TCCAACATGC CCCTCTCCλT CCλCλCCλTC CAACTCCTCT CACTTCCCAC TCλTACλλCC TCCACTAACA AGCTTGTACC CCCλCλCCTλ CCTCTCCTλC CTTCACCACA

>HglAl >ApaLl

0

1690 1700 1710 1720 1730 1740 1750

CTTCCATTCC ACTAACTCTT CCACTTCTCλ CAAACACTCC λCCTACTTCλ ACTTCTACλλ ACAAAACACA CAACCTAACC TCATTCACλλ CCTCλλCACT CTTTGTCACO TCCλTCλλCT TCλλCλTCTT TCTTTTCTCT

>Kap82 >λ«al >Bphl

» ø »

\ 1760 1770 1780 1790 1800 | 1810 1820 • a a a a * • a a v a • • •? •

OCTCCAACTO CACCATTTλC TGCTCCATTT ACACATCλTT TTGAATCCAA TTλλTλλTTA CAACλATCCC CCAOCTTCλC CTCCTAAATO ACGACCTλλλ TCTCTACTAA AACTTACCR AλTTATTλλT GTTCTTACOO

>Sp l

>Rtp(7S24) l >Kap81

>R«pRl > ap(7524) i >P«tl

* » Λ

1830 I 1840 18S0 | lβ60 1870 1880 1890

• a a a * • *ψ a a • • • • •

AAACTCAOCC CCATCCTCAC ATTTAΛCTTT TACATOCCCA ACAACCCCAC ACλλCTCAAA CATCTCCAOT TTTCλCTCCO CCTACCλCTO TλλΛTTCλλλ ATCTAOCCCT TCTTCCOCTO TCTTCACTTT CTACACCTCA

Table 6 (Cont'd)

I 1900 1910 1920 1930 1940 1950 | 1960 y « • • t t • • • • * 4 4 • •

CTCTACAACA ACAACTCAAA CCTCTCCACC λλCTGCTλλλ TTTAOCTCAA ACCAAAAACT TTCACTTλλC CACATCTTCT TCTTCACTTT CCACACCTCC TTCACCATTT AAATCCACTT TCCTTTTTGλ λACTCAATTC

>Avr2

»

>Styl

>Stul >λatl

|| 9?0 1980 2020 2030

VT • • t • « • y β a a a a a a

CCCTACCCAC TTAATCACCA ATλTCAACCT AλTλCTTCTC CACCTAAACC CATCTCAAAC AACATTCATC CCCATCCCTC AATTACTCCT TATλCTTCCA TTATCAACλC CTCCATTTCC CTλCλCTTTG TTCTAACTAC

>BstEl

*

2040 2050 2040 2070 2080 2090 2100

TCTCAATATG CTCATCλCλC λCCCACCATT CTCCAATTTC TCAACACATC CATTλCCTTT TCTCAAACCA ACACTTATAC CACTACTCTC TCCCTCCTλλ CACCTTλλλC ACTTCTCTAC CTAATCCAAA ACλCTTTCCT

>B* * mKl >Batϊl

>sl La.n τlruι 40 a&r y roaotar Ho-MuSV DRA on /aixlaa vLrut 40 MA ■t

2110 2120 2130 3140 " 2150 " I | 3 14!0 " 2170 a a • a a a a a a « / a f a a

TCATCTCAAC ACTλACTTCA TAATTAACTC CTTCCCACTT AAAACATATC λCCλTCCCCT CTCCAATCTC ACTACACTTC TCλTTCλλCT ATTλλTTCλC CλλCCCTCλλ TTTTCTATλO TCCTλOCOCA CACCTTACAC

>B oT22X

>Rsll

>AvaS

Table 6 (Cont ' d)

2180 2190 2200 2210 2220 2240

TCTCACTTAC CCTCTCCAAA CTCCCCACCC TCCCCACCAC CCACAACTAT CCAAACCATC CATCTCAATT ACACTCAATC CCACACCTTT CACCCCTCCC ACOCCTOCTC CCTCTTCATA CCTTTCCTAC CTλCλCTTλA

>N«11

>Ava3

>EcoT22X

a

ACTCAGCAAC CACCTCTCCλ λλGTCCCCAC CCTCCCCACC ACCCACλλCT λTCCλλλCCλ TCCATCTCAA TCACTCCTTC CTCCλCACCT TTCACCCCTC CCλCCCCTCC TCCCTCTTCA TλCCTTTCCT ACCTλCλCTT

2320 2330 2340 2350 2360 2370 2390

TTλCTCACCA ACCλTλCTCC CCCCCCTAAC TCCCCCCATC CCCCCCCTλλ CTCCCCCCλC TTCCCCCCAT AATCACTCCT TCCTATCACC CCGCCCATT ACCCCCCTAC OCCCCCCλTT CλCCCCCCTC λλCCCCCCTA

>KC©1 >8fll >Styi >Bgll

&

2390 2400 2420 2420 2430 | 2440 2450 a «a a a a a • • a a a a * t

TCTCCCCCCC ATCCCTCACT AλTTTTTTTT ATTTATCCAC ACCCCCAGCC CCCCTCCCCC TCTCλCCTλT ACλGCCCCCC TλCCCλCTCλ TTAAAAAAAA TλλλTACCTC TCCCCCTCCC CCCCACCCCC λCλCTCCATA

>Styl

*

>AYΓ2 '

>*tal >B«pMl

Table 6 (Cont ' d )

2460 2470 248 2520

TCCACAACTA CTCACCλCCC TTTTTTCCAO CCCTACCCTT TTCCAAAAAC CTTOCCCTCC ACCTCCλCCC ACCTCTTCAT CACTCCTCCC AAAAAACCTC CCCATCCCAλ AAOCTTTTTC CAACCCCACC TCCACCTCCC

>8cll

CCλTCTCATC AACACACACC ATCAGCATCC TTTCCC ATC ATT CAA CAA CAT CCλ TTC CλC CCλ CCT T CCTλCACTAC TTCTCTCTCC TACTCCTACC AAACOC TAC TAA CTT CTT CTA CCT AAC CTC CCT CCA A

Mat X l a Clu G n Aap Cly Leu Biβ Ala Gly S

>leo52X

>t«gl >taal

Xttl

»

->

2S90| 2600 2610 2620 2630 2640 2650

•• • β β β a a a a a a *

CCO CCC CCT TCG CTC CAC AGO CTA TTC ©CC TλT CλC TCC CCA CAA CAC ACA ATC CCC TCC TC ©CC CCC CCA ACC CAC CTC TCC CλT λλO CCC ATA CTC ACC COT CTT CTC TCT TAC CCO ACC AC rxo Ala Ala Trp Val Ola Arg LM Pba Cly Tyr Aap Trp Ala Cln Ola Thr Xla Cly Cyβ 8a

>Bbol >K4rl >Acyl

*

>Aha2

Table 6 (Cont ' d)

>B»nl

2660 2670 2680 | | 2690 2700 2710 a a * • a ~ • *>• * a t> • a a

CAT CCC CCC CTO TTC CCC CTC TCA CCO CAC CCO CCC CCO CTT CTT TTT CTC AAC ACC CλC CTA CCC CCC CAC AAG CCC CAC ACT CCC CTC CCC CCC CCC CAA CAA Aλλ CAC TTC TCC CTC C

Aap Ala Ala Val Pha Arg Lau Sar Ala Cln Cly Arg Pro Val Uu Pha V«l Lya Thr Aap

>Cfrl

* 2720 2730 2740 2750 2740 2770

TCC GOT CCC CTC AAT CAA CTC CAC CAC CAO CCA CCC CCC CTA TCG TCC CTC CCC ACC ACC C ACC CCA CCC CλC TTλ CTT CλC CTC CTG CTC CCT CCC CCC CλT λCC λCC CλC CCC TCC TCC C Sar Cly Ala Uu Aan Clu Uu Gin Aap Glu Ala Ala Arg Uu Sar Trp Uu Ala Thr Thr C

>Fapl

>Aoal >Tthll X >P(J12 >Pvu2 >HglAl

0 0

>Katl >Rtp42 ø ø 80 |2740 i i 290θ| 2820 2820 2830 2840

CTT CCT TCC CCA CCT CTC CTC CλC CTT CTC ACT CAA CCC CCA ACC CAC TCC CTC CTA TTC CC CAA CCλ ACC CCT CCλ CAC CAC CTC CAA CAC TCA CTT CCC CCT TCC CTC ACC CAC CλT AAC CC Val Pro Cya Ala Alt Val Uu Aap Val Val Thr Clu Ala Cly Arg Aap Trp Lou Uu Uu Cl

>IβtTl >X3vθ2 >Hphl '

28S0 I 28(0 I 3870 2880 2890 2900

• a «y a T a a a a a a a

CAA CTO CCO OCC CAO CAT CTC CTC TCA TCT CAC CTT CCT CCT CCC CAO Aλλ CTA TCC ATC AT CTT CAC CCC CCC CTC CTA CAO CAC ACT ACA CTC CAA CCA CCA CCO CTC TTT CΛT AGO TAG TA Clu Val Pro Cly Ola Aap Uu Uu Sor Sor Bia Uu Ala Pro Ala Cla Lya Val Sar Xla Kβ

>BapKl

Table 6 (Cont ' d)

2910 2920 2930 3940 2950 I 29(0

CCT CAT CCλ ATC CCC CCC CTG CAT ACC CTT CAT CCO CCT ACC TCC CCA TTC CAC CAC CAA C CCA CTA CCT TλC CCC CCC CλC CTA TGC CAA CTA GCC CCλ TCG ACC CCT AAC CTC CTG CTT C

Ala λap λla Mat Arg Aro. Uu Hla Thr Uu λap Pro λla Thr Cyβ Pro Phe Aap Hla Cln λl

>Raal

>Hglλl

*

>Maa2 XTfrlOI

2970 2960 2990 3000 I 3010 3020 303 λλλ CAT CCC ATC CAC CCλ CCλ CCT λCT CGC λTO CAA CCC CCT CTT CTC CλT CλC CλT CλT CT TTT CTA CCC TAC CTC CCT CCT CCλ TCA CCC TAC CTT CCC CCλ Cλλ CλC CTA CTC CTA CTA Cλ Lya Ria Arg Xla Clu Arg λla λrg Thr Arg Mat Clu λla Cly Uu Val λap Cln Aap λap U

>Sphl

>8an2 >8ιaK2 >KapMl ø • *

3040 3050 3040 3070 3080 I 3090

CλC Cλλ CAC CλT CAC CCC CTC CCC CCλ CCC Cλλ CTC TTC CCC λCG CTC AAC CCC CCC ATC CC CTC CTT CTC CTA CTC CCC CAC CGC CCT CCC CTT CAC AAG CCC TCC CAC TTC CCC CCC TAC CC λap Clu Clu Mis Cln Cly Uu λla Pro λla Clu Uu Pha λla λrg Uu Lya λla λrg Mat Pr

>Xho2 >Mcol JC

* 0

>BstYl >Styl >E

* *

3100 J 3110 |3120 3130 3140 3150

CλC CCC CAC CλT CTC CTC CTC ACC CAT CCC CλT CCC TCC TTC CCC AAT ATC ATC CTC Cλλ λλ CTO CCC CTC CTA CAC CAC CλC TOO CTA CCC CTA CCO ACC AAC CGC TTλ TλC TλC CAC CTT T Aap O y Olu Aap Uu Val Val Thr Bia Oly λβp λla Cyβ Uu Pro λaa Xla Hot Val Oiu Aβ

3140 3170 3210

CCC CCC TTT TCT CCλ TTC λTC CλC TCT CCC CCC CTC CCT CTC CCC CλC CCC TλT CλC CλC CCC CCC λλλ ACA CCT AAC TAG CTC ACλ CCC CCC CλC CCλ CAC CCC CTC CCC λTλ CTC CTC Cly λrg Pha Bar Cly Phe lie λβp Cyβ Cly λrg Uu Cly Val λla λβp Arg Tyr Cln λ β p

20 3230 3240 3250 3260 3270 328

CCC TTC CCT ACC CCT CAT ATT CCT CAA CAC CTT CGC CCC CAA TCC CCT CAC CCC TTC CTC C CCC λAC CCλ TCC CCλ CTλ TAA CGA CTT CTC CAA COG CCC CTT ACC CCλ CTC CCC AAO CAC Ala Uu Ala Thr λrg λβp 11a λla Clu Glu Lau Gly Cly Clβ Trp λla λap λxg Pha Uu V

3290 3300 3310 3320 3330 3340

CTT TλC COT λTC CCC CCT CCC CλT TCC CλC CCC λTC CCC TTC TλT CCC CTT CTT CλC CλC T CAA ATC CCA TAG CCC CCλ CCC CTλ ACC GTC CCC TAC CCC λAC ATA CCC Cλλ CAA CTC CTC λ Lou Tyr Cly Xla λla λla Pro λβp Sar cla λrg Xla λla Phe Tyr λrg Uu Leu λβp Clu P

>Plel

>TnS_ORA_βftd/ »θ-MuLV_DKA_βtert

33SO 33(0 3370 3380 3390 3400 2410 34

TTC TCA CCCOCACTC TCCCCTTCCλ TλλλλTλλλλ CATTTTλTTT λCTCTCCλCλ AAAACCCCCC λATCAAAC AAC λCT CCCCCTCλC ACCCCAACCT ATTTTλTTTT CTAAAATAΛA TCACλCCTCT llllCCCCCC TTλCTTTC Pha tnd>

>λf!3

>Rhel

3430 3440 I 3450 34(0 3470 3480 3490

CCCλCCTCTλ CCTTTCCCλλ CCTλCCTTλλ GTλλCGCCλT TTTCCAACGC λTCCλλλλλT λCATλλCTCλ CCCTCCλCAT CCAAACCCTT CCATCCλλTT CATTCCCCTλ λλACCTTCCC TλCCTTTRA TCTATTCACT

a

>Pvu2 >E oRS

3500 3510 3520 3530 | 3540 3550 | 35(0

• a a a a a a a * a a a a * / a

CλλTλCλCλλ CTTCAGλTCλ ACCTCλCCλλ CACλTCCλλC ACCTCλλTλT CCCCCλλλCA CCATATCTOt CTTλTCTCTT CAACTCTλCT TCCACTCCTT CTCTλCCTTC TCCλCTTλTλ CCOCCHlUI CCTλTλCλCλ

>AlwNl >Pvu2 >EcoRS

3570 I 3S80 3S90 3600 3610 3630 3630

• • y • • • • a * 4 y β • 4 ) β y«

CCTAACCACT TCCTCCCCCC CCTCACCCCC λACAACACλT CCAACACCTC AATATCCCCC AλλCACCλTλ CCATTCCTCA ACCACGCCCC CCACTCCCCC TTCTTCTCTA CCTTCTCCAC TTATλCCOCC TTTCTCCTλT

>λlwNl

3640 3650 3660 3670 3680 3690 3700

TCTCTCCTλλ CCΛCTTCCTC CCCCGCCTCA CCCCCAACAA CACATCCTCC CCACATCCCC TCCACCCCTC ACλCACCATT CCTCAACCAC CCCCCCCACT CCOCCTTCTT CTCTACCAOG CCTCTAOCCC λCCTCCCCλC

λCCACTTTCT ACλCAλCCλT CACATCTTTC CλCCCTCCCC CΛAGCACCTG AAATCACCCT CTCCCTTλTT TCCTCAAACA TCTCTTCCTA CTCTACAAAC CTCCCλCCCG CTTCCTCCλC TTTACTCCCλ CACGCλλTλλ

ø

3780 3790 3800 3810 3840

TCλλCTAACC λλTCAOTTCO CHCICOCTt CTCTTCCCCC CCTTCTCCTC CCCCAOCTCλ λTλAAACλOC λCTTCλTTCC TTACTCλλCC CλλCλCCCλλ CλCλλCCCCC CGAACλCCAG CCCCTCCACT TATTTTCTCC

>Banl

>Baa2 >λap718

>Bbal >*l.lj

Table 6 (Cont'd)

H rl >S«al

>B«nl >Xa*l

>λoul > λIcyl >λv -alI >Rpnl

. |. . |

>Ban2 >Aval >Ah*2 >τthl l lX >λgul >Rβal

| . . J

I 3650 j 3860 3870 3680 3890 J I 3900 3910 y • • y« y« y • * * • • y y* y y

CCACAACCCC TCACTOCCCC CCCCACTCCT COGATTCλCT CλCTCCCCCC CCTλCCOCTC TλTCCAATAA CCTCTTOCCC AGΪCλCCCCC COCCTCACCA CCCTAλCTCA CTCACCCCCC CCATCCCCAC λTAGGTTATT

>Styl

3920 3930 3940 39SO 39(0 3970 3960 λCCCTCTTCC ACTTCCATCC CACTTCTCCT CTCOCTCTTC CTTCCCACCC TCTCCTCTCA CTCλTTCACT TOCCACAACC TCAACCTACC CTCAACACCA CACCCACAAG CAACCCTCCC ACλGCλCACT CACTAACTCλ

3990 4000 4010 I 4020 4030 4040 4050

ACCCCTCACC CCCCCTCTTT CATTTCCCCC CTCCTCCCCC ATCGCCλCλC CCCTCCCCAC CCλCCACCCλ TCGGCλCTCC CCCCCACλλλ CTλλλCCCCC CACCAGGCCC TAGCCCTCTC CCCACCCCTC CCTCCTCCCT

>Rρhl >Rphl >RβpBl

>Mo-MuLV_DXλ end/plιeaid p8R322 X>Kλ, ιa> etart

40(0 4070 I 40(0 4090 4100 4210 4120 a a a a y a a a a a a y a a τ a «a

CCCλCCACCO CCACCTλλCC TCGCTCCCTC CCCCCTTTCC CTCλTCλCOC TCλAλλCCTC TCλCACATCC CCCTCCTCCC CCTCCATTCC λCCCλCCCλO CCCCCAAAGC CλCTλCTCCC λCTTTTCCλC λCTCTGTλCC

>8gal

4130 4140 4150 41(0 4170 I 4180 4190

ACCTCCCGCλ CλCCCTCλCλ CCTTCTCTCT λλOCGCATCC CCCCλCCλCλ CAACOCCCTC λCCCCCCCTC TOCλCGCCCT CTCCCλCTCT CCλλCλCλCλ TTCCCCTλCO CCCCTCCTCT CTTCCCCCAO TCCCCCCCM

>HepB2 >TthlllX >Maa2 >Accl

Table 6 (Cont ' d)

I 4200 4210 4220 4230 | 4240 4250 | 4260 λCCCCCTCTT CCCCCCTCTC CCCCCCCACC CATCACCCAC TCAOCTACOC ATλCCCCλCT CTλTλCTCCC TCCCCCACAλ CCCCCCACAC CCCCCOCTCC CTACTCCCTC λCTCCATOCC TATCCCCTCA CλTλTCACCC

>H Lλl

4270 4280 4320 4320 4330

TTAACTATCC CCCλTCλCλC CACATTCTλC TCλCλCTCCλ CCATλTCOGC TCTCAAATAC CCCACACATC AATTCATACC COGTACTCTC CTCTAλCAIC ACTCTCACCT CCTλTλOCCC λCλCTTTλTC CCCTCTCTλC

* *

4340 4350 4360 4370 | 4380 4390 4400 • • • v • • • v * • a a • •

OCTAAGCλCλ AAATλCOCCλ TCACCCCCTC TTCCCCTTCC TOCCTCλCTC λCTCCCTCCC CTCCCTCCTT CCATTCCTCT TTTATCCCCT λCTCCCOCλC λλCCCCAλCC ACCCλCTCAC TCACCCλOCC CλCCCλCCλλ

4410 4420 4430 4440 4450 4460 4470

CGGCTCCCCC CACCCCTλTC λCCTCλCTCλ AACCCCCTAA TACCCTTATC CACACλλTCλ CCCCATλλCC CCOCλCCCCC CTCCCCATAC TCCACTCλCT TTCCCCCATT λTGCCAATAC CTCTCTTACT CCCCTATTCC

>Rap(7S24)l >RβpRl

>λfl3

*

4480 4490 4500 4510 4520 4530 4540

CλCGAλλCλλ CλTCTCλCCλ λAλGCCCλCC AAAACCCCAC CAACCCTλλλ λλCCCCGCCT TCCTCGCCTT CTCCTTTCTT CTACACTCCT TTTCOCCTCC TTTTCOCCTC CTTGCCATTT TTCCCCCCCA λCCλCCCCλλ

>Rgal

4550 45(0 4570 4580 4590 | 4(00 4(10

Table 6 (Cont ' d)

TTTCCATACC CT«»CCCCC CTCACCACCλ TCACAAAAAT CCACCCTCAA CTCACACCTC CCCAλλCCCO AAACCTATCC CACCCCCCCC CACTCCTCCT λCTCTTTTTλ CCTCOCACTT CACTCTCCλC CCCTTTCCCC

4620 4630 4640 4650 4660 4670 4680

ACAOCACTAT AAACATACCA CCCCTTTCCC CCTCCAAGCT CCCTOCTCCC CTCTCCTCTT CCCACCCTCC TCTCCTCATλ TTTCTATCCT CCCCAAACCC CCACCTTOCA CCCACCACCC CλCλGCACλλ CCCTCOCAOC

>Haa2

4690 4700 4710 4720 4730 4740 4750

CCCTTACCOC ATACCTCTCC CCCTTTCTCC CTTCCCGλλG CCTCCOCCTT TCTCATλCCT CλOCCTCTλG CCCAATCCCC TATCCACACS CCCAλλCλCC CAACCCCTTC CCACCGCCλΛ ACλCTATCCλ CTCCCλCATC

>Rglλl

4760 4770 4780 4790 4810 4820

CTλTCTCACT TCCCTCTACC TCCTTCCCTC CAACCTGGCC TGTCTGCACC AλCCCCCCCT TCλCCCCCλC CλTAGλCTCλ ACCCACATCC ACCAACCCAC CTTCGACCCC ACλCλOCTCC TTCCCCCCCA λCTCCGGCTC

>NapB2 >Plal

I 4830 4140 4850 4860 4470 4880 4890 a a • a a t t a «• » • t • • %

OCCTGCGCCT TλTCCCCTλλ CTATCCTCTT CAGTCCAACC CCCTAACACA CCλCTTλTCC CCλCTCCCλC CCCACCCCCλ ATACCCCATT CATλCCλCAA CTCACCTTCC CCCATTCTCT CCTCAATλCC CCTGλCCCTC

>λlwNl

14900 4910 4920 4930 4940 4950 4960

CACCCλCTCG TAACAGCATT ACCλCλCCCλ CCTATCTAOC CCGTCCTACλ CλCTTCTTCλ λCTCGTGCCC CTCGCTCλCC λTTCTCCTAA TCCTCTCCCT CCATACATCC GCCAOCλTCT CTCAAGAACT TCλCCACCCC

4970 4980 4990 5000 5010 S020 5030

TλλCTλCGCC TACACTλCAA CCACACTATT TCCTATCTCC CCTCTCCTCλ ACCCACTTAC CTTCCCλλλλ ATTCλTCCCC λTCTCλTCTT CCTCTCATAA λCCλTλCλCC CCλCλCCλCT TCCCTCAATC CAACCCTTTT

>R β pB2

λCACTTCCTA CCTCTTCATC CCCCAAACAA ACCACCCCTC CTACCCCTCC T1TTI ITCTT TCCAACCACC TCTCAACCAT CCACλACTAC COCCTTTCTT TCCTCCCCλC CATOCCCACC AAAAAAACAA ACCTTOGTCC

>Xho2 >8atTl

>Bβtri >Xho2 >Rgal

* * *

5110 5120 I S130 | S140 5150 5160 5170 λCATTλCCCG CAGAAAAAAλ CCATCTCλλC AAGATCCTTT CAT 1T1TCT λOCCCGTCTC λCCCTCλCTC TCTAATCCOC CTCTTTTTTT CCTAGACTTC TTCTACCλλA CTλCλAAAGλ TGCCCCAGAC TCOCACTCAC

>BetYl a\

>Xho2 >BatXl >Dral

>Mae2 >BapHl >Hphl >Xho2 >Aha3

* a A I * •

5180 I 5190 5200 S210 | S220 5230 5240

* • y • • t • • • *y y • • • • y*

CλλCCAλλAC TCACCTTλAC OCATTTTOCT CATCλCATTλ TCAAAAλGGλ TCTTCACCTA CλTCCTTTTλ CTTGCTTTTC λCTCCAATTC CCTAAAλCCλ CTACTCTAλT λCTTTTTCCT λCAACTCCAT CTλCCλAAAT

>Dral

>Aha3

5250 5240 S270 S2I0 5290 5300 5310 a t a y β t t • t t) • a a a a

AATTAAAAAT CλλCTTTTλλ ATCAATCTAA ACTλTλTATC λCTλλλCTTC CTCTCλCλCT TλCCλλTCCT TTλATTTTTλ CTTCAAAATT TACTTACATT TCλTATλTλC TCATTTCλλC CACλCTCTCA λTCCTTλCCA

ø ø

5320 I 5330 S340 S3S0 53(0 | 5370 5380 a a y * a a a % t a β y β a a a

TλλTCλCTCλ CCCACCTλTC TCλCCCλTCT CTCTλTTTCC TTCλTCCATλ CTTCCCTCλC TCCCC TCCT λTTλCTCλCT CCGTCCATλC λCTCCCTλCλ CACλTλλλCC AACTACCTAT CλλCCCλCTC λGCCCCACCλ

>Bphl

1390 5400 5410 5420 S430 5440 | S4S0 a a a a a a a a • a a y a a

CTACATAACT λCCλTλCCGG ACCCCTTλCC ATCTCCCCCC λCTCCTCCλλ TCλTλCCCCC λCλCCCλCCC CATCTλTTCλ TCCTλTCCCC TCCCCAATCC TACλCCCCCC TCACCAOCTT ACTλTCGCCC TCTCCCTCCC

-CfrlOl >Bgll

* »

I 54(0 5470 5480 5490 j 5500 5510 5520

T • • • * a • • y a a • * a a

TCλCCGCCTC CAGλTTTλTC ACCλλTλλλC CACCCλCCCC CλλGCCCCCA CCCCACλλCT CCTCCTCCλλ λCTCCCCCλC CTCTAAATAC TCCTTATTTC CTCCCTCCGC CTTCCCCCCT CCOCTCTTCλ CCACCλCCTT

Table 6 (Cont ' d)

5530 5540 | 5550 5560 5570 5580 5590

CTTTλTCCOC CTCCATCCAC TCTATTAATT CTTCCOCCCA λCCTλCλCTλ λCTλGTTCCC CλCTTλλTAO CλλλTACCCG CAGCTACCTC AGλTAATTAA CAλCCCCCCT TCCλTCTCλT TCλTCλλCCC CTCλλTTλTC

5630 5640 5650 56 ( 0 a y* a a • y • a * a a * a t)

TTTCCCCλλC CTTCTTCCCA TTCCTCCλCC CATCCTCCTG TCλCGCTCCT CCTTTCCTAT CCCTTCATTC λλλCGCCTTC CλλCλACCCT AACCACCTCC CTλCCACCAC λCTCCCλCCλ CCAAACCATA CCCλλGTλλC

5(70 5(80 5(90 5700 5710 5720 5730 λCCTCCCCTT CCCAACCATC AACCCCACTT λCATCATCCC CCATCTTCTC CλλλλλλGCC CTTλGCTCCT TCCλGGCCλλ CCGTTCCTAC TTCCCCTCAλ TCTλCTλCCC GGTλCλλCλC CTTTTTTCCC CAATCCACCλ

5770 5780 5790 5800

TCCCTCCTCC CATCCTTCTC λCλλCTAAGT TCCCCGCλCT CTTATCλCTC ATCCTTλTCC CλGCλCTGCA λCCCACCλGG CTAGCAACλG TCTTCλTTCλ λCCCCCGTCλ CAATACTCAC TACCAATACC CTCGTGλCCT

*

>Scal >Rphl

5810 5820 5830 5840 5850 | 58(0 5870 a * a a • • • • * • y * y* % %

TλATTCTCTT λCTCTCATCC CATCCCTAAG λTCCTTTTCT CTCλCTCGTO ACTλCTCλλC CλλGTCλTTC ATTλλGAGλλ TCACλCTλCC CTλCCCλTTC TACCλλλλCλ CλCTCλCCλC TCλTCλGTTO CTTCλCTλλO

Table 6 (Cont ' d)

>Hlne2 >Klnd2

>λcyl

* *

S860 5890 S90O 5910 5920 5930 5940

TCλCAATACT CTATCCCCOC ACOCλGTTCC TCTTCCCCCC OCTCAΛCλOO CCλTAATACC GOGCCACλTλ ACTCTTλTCA C-ATACCOOCC TCOCTCAAOC λCλλCCCCCC CCλCTTCTCC CCTλTTλTCC CCCOCTCTλT

>λha3 >Mβa2

*

>Dral >Hg l >XΛΛ1

5950 59(0 5970 I 5980 5990

CCAGλλCTTT λAAλCTCCTC ATCλTTCCλλ λACCTTCTTC CCCCCCλλλλ CTCTCAACGλ TCTTλCCCCT CCTCTTCλλλ TTTTCACCλC TACTAλCCTT TTCCλλCλλC CCCCCCTTTT CACλGTTCCT λCAATCCCCA

>Xhθ2 >Hgiλl

>Bβtn >λpaLl >Rphl a\ *

I (020 6030 6040 (050 (0(0 I 4070 (080

CTTCλCλTCC λCTTCCλTCT λλCCCACTCO TCCACCCλλC TCATCTTCAC CλTCTTTTAC TTTCλCCλOC CλλCTCTλCG TCλλCCTλCλ TTCGCTCACC ACCTCCCTTC λCTλCλλGTC CTACAAAATC λλλCTCCTOΘ

>Bphl

*

(090 (100 (110 (120 (130 4240 4150

CTTTCTCGGT CACCλλλλλC λCCλλCCCAλ λλTCCCGCλλ AλAλCCCλλT AACGCCCACA CCCλAATCTT CλλλCACCCA CTCCTTTTTC TCCTTCOCTT TTλCCCCCTT TTTTCCCTTλ TTCCCCCTCT CCCTTTACAA

>5apl >BβpRl

0

(1(0 (170 ( 180 (190 (200 €210 (220

Table 6 (Cont ' d)

CAATACTCAT λCTCTTCCTT TTTCAATATT ATTCAACCAT TTATCACCCT TATTCTCTCA TCACCCCATA CTTATCACTA TCACAACCAA AAACTTATAA TAACTTCCTA AATACTCCCA ATAACACACT ACTCCCCTλT

(230 (240 (250 62(0 6270 6280 6290

CATATTTCAA TCTATTTACA AAAATAAACA AATACCCCTT CCGCCCACAT TTCCCCCAAA ACTGCCACCT CTATAAACTT ACATAAATCT TTTTATTTCT TTATCCCCAA CCCCCCTCTA AAGCCCCTTT ICACCCTCCA

>λat2

6360

CλCCTCTλλG λλλCCΛTTAT TλTCλTCλCA TTλλCCTλTλ λλAATλCCCC TATCλCCλCC CCCTTTCCTC CTGCACλTTC TTTCCTAλTA λTλCTλCTCT AATTCCATλT TTTTλTCCCC λTλCTCCTCC CCCAλλCCλO

a TTCAA

AACTT

cysoa which do not cut LXSXX2XL2 t

To generate the LXSN-RI-IL2 retroviral vector, 10 micrograms of pLXSN-RI-IL2 DNA was transfeeted 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 μg/ml). The cells were grown at 37°C in 7% C0 2 atmosphere. The selection medium was changed every 5 days until colonies appeared. On day 14, twenty colonies were selected, expanded and tested for viral production by standard methods (Xu et al., Virology 171:331-341, 1989). Briefly, supernatants were harvested from confluent culture dishes, passed through a .45 μm filter, diluted with DMEM with 10% FCS and utilized to infect NIH 3T3 cells in the presence of 8 μg/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 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.

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EXAMPLE IV

RETROVIRAI, 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 (Figure 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 μg/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.

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Table 7

Comparison of IL-2 expression by fibroblasts transduced with different IL-2 vectors.

nσ IL-2 Units IL-2

Fibroblast Vector per 10 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% 4

Human LXSN (Control) 0.7 ±29% 1 LNCX-IL2 159.5 ±17% 319 LXSN-IL2 25.5 ±15% 51 DC/TKIL-2 3.0 ±10% 6

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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 x 10 6 primary fibroblasts from a 12 mm 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 Gl 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.

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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 GTl cells, a primary human fibroblast culture expressed 15-fold more IL-2 under 100 μg/ml G418 selection than under 25 μg/ml G418 selection. Several other primary fibroblast lines have also been transduced with our vectors and are currently growing under G418 selection.

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Table 9

Effect of G418 concentration on IL-2 expression by GTl cells transduced with LXSN-IL2.

Selection dose ng IL-2 secreted of G418 per 10 6 cells per day *

25 μg/ml 1.0 ± 10% 50 μg/ml 3.0 ± 6%

100 μg/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 μM calcium ionophore (CI) (A23187) free acid) plus 17 nM phorbol 12- yristate 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 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 6 cells/24 hours. This was lower than the PMA/CI stimulated culture, presumably because PMA/CI induced a nonspecific response

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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 secreted

Cells per 10 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

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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 VIIfa)

EFFECT OF FIBROBLAST MEDIATED CYTOKINE GENE THERAPY ON TUMOR IMPLANTATION

Mice were injected subcutaneously with mixtures of 5 x 10 4 CT26 cells and 2 x 10 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 (Figures 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.

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Table 11

Effect of IL-2 modified fibroblasts on tumor establishment and development. 2 X 10$ fibroblasts mixed with 5 X 104 CT26 tumor cells at time of mjection.

* 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 2 , compared to 68 and 7 mm 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 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 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 Figure 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 x 10 ε modified fibroblasts mixed with 1 x 10 s live tumor cells the results became more striking (see Figures 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

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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.

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Table 12

Effect of IL-2 modified fibroblasts on tumor establishment and development. 2 X 10<5 fibroblasts mixed with 1 X 10* CT26 tumor cells at time of injection.

CD

C OP

H m D -E rn m

H

* 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.

*** 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 x 10 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 x 10 6 CT26 tumor cells modified with the DCTK-IL2 vector with 1 x 10 5 unmodified tumor cells. Figures 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 2 , compared to 620 mm 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.

SU5 T c HEET

Table 13

CD Effect of IL-2 modified cells on tumor establishment and development. 2 X 106 DCTK-IL2-modified CT26 tumor cells mixed with 1 X 105 CT26 cells compared to 2 X 106 DCTK-IL2-modified fibroblasts mixed with 1 X 105

V CT26.

H C -\ m

CD I m

21

Mean tumor size is for 4 weeks, me last timepoint at which tumors were measured.

EXAMPLE VIKbi

EFFECT OF FIBROBLAST MEDIATE CYTOKINE GENE THERAPY ON SYSTEMIC ANTI-TUMOR IMMUNITY

Groups of Balb/c mice were immunized with 2.5 x 10 5 irradiated tumor cells either alone or mixed with 2 x 10 6 transduced or unmodified fibroblasts, and challenged one week later with 5 x 10 4 live tumor cells in the opposite flank. These results (Figures 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.

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Table 14

Effect of IL-2 modified fibroblasts on induction of sytemic anti-tumor immunity. Mice immunized with 2 X 106 fibroblasts mixed with 2,5 X 105 irradiated CT26 tumor cells 7 days prior to challenge with 5 X 104 fresh tumor cells,

0 C

C m > x m rπ

H

* 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 Figures 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 2 , compared to 180, 170, and 140 mm 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 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 Figure 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).

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Table 15

Effect of IL-2 modified fibroblasts on induction of sytemic anti-tumor immunity. Mice immunized with 2 X 106 fibroblasts mixed with 2.5 X 10 irradiated CT26 tumor cells 14 days prior to challenge with 5 X 104 fresh tumor cells.

CD

C TO

H

C m D x m m

H

* 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.

- ::, T|Ti ΪTC ajppT

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., i_8_.853-864 (1988).

5. Lotze, M.T. et al., JAMA, 256.:3117-3124 (1986).

6. Pizza, G. et al., Lymphokine Research, 2*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, 6fJ: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).

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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., 3 :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, 121:331-341 (1989).

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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.

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