KR100537731B1 - Pharmaceutical Composition comprising Interleukin-2 gene transferred lymphokine activated killer cells - Google Patents

Abstract

The present invention provides a pharmaceutical composition comprising lymphokine active killer cells (I-LAK cells) transfected with a recombinant vector to which the interleukin-2 gene is operably linked. Lymphocine-activated killer cells I-LAK according to the present invention secrete low concentrations of interleukin-2 and have the activity of selectively killing cancer cells or virus-infected cells without damaging normal cells, thereby treating cancer and treating viral infections. Very useful for

Description

Pharmaceutical Composition comprising Interleukin-2 gene transferred lymphokine activated killer cells

The present invention relates to lymphokine active killer cells (I-LAK cells) transfected with a recombinant vector to which the interleukin-2 gene is operably linked. Specifically, the present invention I transfected lymphokine active killer cells (LAK cells) prepared by separating from the peripheral blood of a mammal with a recombinant vector operably linked to the interleukin-2 gene introduced I It relates to a -LAK cell, a method for producing the same and an anticancer or antiviral pharmaceutical composition comprising the same. I-LAK cells according to the present invention continuously secrete low levels of interleukin-2 and have the activity of selectively killing cancer cells or cells infected with viruses.

Lymphokine activated killer cells (hereinafter referred to as LAK cells) are mainly used for adoptive immunotherapy for the treatment of malignant melanoma and colorectal cancer. Adoptive immunotherapy using LAK cells stimulates and activates lymphocytes isolated from blood collected from cancer patients with a substance called Interleukin-2 (hereinafter referred to as IL-2) to produce LAK cells. The produced LAK cells are put back into the body of cancer patients.

Such immunotherapy is a treatment that has been developed in recent decades, and focuses on restoring the body's original immune function or enhancing the resistance of patients to cancer. In general, it is known to be particularly effective in cancers with relatively high immunity, such as malignant melanoma.

  In addition to LAK cells, immunotherapy agents used in immunotherapy include BCG, interferon, interleukin-2, tumor necrosis factor, unicellular antibody, fish vinyl, helix, and the like. BCG, a type of tuberculosis bacterium, was used in the early days of immunotherapy, and is known to enhance malignant melanoma by enhancing the function of phagocytic cells that eat cancer cells and enhancing lymphocyte immunity. Inteferon affects not only cancer cells but also normal cells, causing many side effects, which are rarely used at present.

Methods of making LAK cells are described, for example, in US Pat. No. 4,849,329 to Leung et al.

U.S. Patent No. 5,108,760 (Irr et al.) Describes LAK cells having increased activity of amino acid amide treatment on peripheral blood mononuclear cells and pharmaceutical compositions for the treatment of cancer comprising the same.

US Patent No. 4690915 (Rosenberg; Steven A.) discloses a method of treating cancer by co-administration of LAK cells with IL-2.

LAK cells, on the other hand, can be used not only for the treatment of cancer but also for the treatment of HIV infection, which is at the stage of in vitro research. It has been demonstrated that LAK cells are effective in removing HIV-infected cells in vitro (Tyler DS et al., J. Surg. Res., 79, 115-120 (1998); Wang L., et al. Virology, 241, 169-180 (1998).

Although the treatment of HIV infection has advanced rapidly in recent years, it has not yet cured the infection. Among the therapies developed so far, the standard treatment for HIV infection is a combination of three or more antiretroviral agents, called highly active antiretroviral therapy (HAART) (Hammer S. et al., N. Engl. J. Med., 337, 725-733 (1997); Staszewski S., N. Engl. J. Med., 341, 1865-1873 (1999). However, this therapy is limited in treating HIV infection due to the disadvantage that anti-retroviral agents do not work on HIV cells in the latent period and do not penetrate the human anatomical boundaries (cerebrovascular barrier, testicular vascular barrier, etc.) in sufficient concentration. have. Another treatment for HIV is the administration of IL-2 together with the combination therapy of the antiretroviral agent (Chun TW et al., Nature Med., 5 (6), 651-655 (1999)). However, these therapies also have limitations in curing HIV infection.

In the treatment of cancer and HIV infection, the following problems must be overcome for clinical application of LAK cells: Typically, in immunotherapy with LAK cells, LAK cells are required to ensure that the LAK cells continue to survive in the human body. And high concentrations of IL-2. High concentrations of IL-2 have been reported to activate the viral virus in the blood in the blood (viral blips), which in turn increases latent cells (Ramratnam B et al., Nat Med, 6 (1), 82-). 5 (2000)). In addition, continuous administration of high concentrations of IL-2 from the outside has a problem that various side effects occur, including cytokine leak syndrome (cytokine leak syndrome).

The present inventors transfected LAK cells with a recombinant vector operably linked to the IL-2 gene while studying anticancer and antiviral agents to prepare I-LAK cells into which the IL-2 gene was introduced, and the I-LAK cells were The present invention was completed by confirming the ability to continuously secrete low concentrations of IL-2 and to selectively kill cancer cells or virus-infected cells without damaging normal cells.

The present invention provides anti-cancer or antiviral lymphokine active killer cells, ie I-LAK cells, transfected with a recombinant vector to which the IL-2 gene is operably linked.

Preferably, I-LAK cells of the invention are transfected with a retroviral vector to which the IL-2 gene is operably linked. More preferably, I-LAK cells of the invention are transfected with the retroviral vector LNC / IL-2 / IRES / TK.

In addition, the present invention

(a) bleeding peripheral blood from a mammal and removing red blood cells from the peripheral blood to obtain a lymphocyte-containing leukocyte fraction,

(b) it is cultured in a culture medium containing IL-2 to produce LAK cells,

(c) transforming the resulting LAK cells with a recombinant vector comprising an IL-2 secretory gene and culturing them under appropriate media and conditions to differentiate the cells,

(d) selecting and recovering cells having IL-2 secretion ability from the cultured cells, and providing a method for producing I-LAK cells into which the IL-2 gene has been introduced.

The present invention also provides a pharmaceutical composition for treating a cancer or viral infection disease comprising an amount of said I-LAK cells sufficient to elicit an immune response.

In one aspect of the invention, the invention provides anti-cancer or antiviral lymphokine active killer cells (I-LAK cells) transfected with a recombinant vector to which the IL-2 gene is operably linked.

I-LAK cells of the present invention continuously secrete a certain amount of interleukin-2 to maintain a constant low concentration of interleukin-2 in vivo without a separate IL-2 treatment and to cancer cells or viruses without damaging normal cells Has the property of selectively killing infected cells.

Therefore, when the I-LAK cells of the present invention are administered to mammals, IL-2 concentrations can be kept constant at low concentrations by IL-2 secreted from I-LAK cells, thereby preventing cancer cells without additional IL-2 treatment. Or cytotoxicity, which selectively kills cells infected with the virus.

LAK cells are nonspecific immune effector cells and possess the ability to lyse virus infected cells and various types of transformed cells through a non-MHC-limiting mechanism.

LAK cells are prepared by culturing peripheral blood mononuclear cells with interleukin-2 and can be used to treat cancer, viruses and other infectious diseases, autoimmune diseases, and the like.

US Pat. No. 4,690,915 describes that the combination of LAK cells and IL-2 is effective in treating human melanoma, lung cancer, kidney cancer, colon cancer and rectal cancer. Rayner et al., Cancer, 55, 1327-1333 (1985) also describe various tumor killing activities, including colon cancer, pancreatic cancer, esophageal cancer, etc. of LAK cells.

IL-2 is a potent immunomodulatory cytokine, described for the first time as a lymphokine capable of promoting long-term proliferation of activated T cells in vitro (Morgan et al., Science, 193, 1007-1008 (1976) ). IL-2 is produced by CD4 T lymphocytes, and much less is produced by CD8 T lymphocytes. IL-2 functions as an autocrine growth factor that acts on the same cells that produce it. IL-2 also acts as a paracrine growth factor because it affects adjacent T lymphocytes, including CD4 and CD8 cells. IL-2 is also known to modulate various immune functions, including affecting cytotoxic T cells, NK cells and activated B cells, and to enhance cytolytic function by producing LAK cells.

IL-2 introduced into LAK cells in response to the object of the present invention is IL-2 having natural IL-2 activity that allows LAK cells to continuously survive in the human body. CDNA encoding human IL-2 has been cloned (Taniguchi et al., Nature, 302, 305 (1983)), from which protein sequences have been estimated. In eukaryotic cells, IL-2 is a precursor poly consisting of 153 amino acids. Produced as a peptide, 20 of these amino acids are removed, resulting in mature IL-2. Using molecular biological methods, active recombinant human IL-2 was isolated from E. coli (Rosenberg et al., Science, 223, 1412 (1984)), insect cells (Simth et al., Proc. Natl. Acad. Sic). USA, 82, 8404 (1985)) and mammalian COS cells (Taniguchi et al., Nature, 302, 305 (1983)).

Using this known technique, the IL-2 gene introduced into LAK cells of the present invention can be obtained from any suitable source, including known recombinant IL-2 genes. Specifically, the IL-2 gene includes natural and recombinant IL-2 and their biologically functional equivalents, and the biological equivalents are, for example, recombinant IL-2 muteins and natural IL-s described in US Pat. No. 4,518,584. Polypeptides having the same or very similar biological activity, such as recombinant IL-2 proteins with methionine replacing the NH ₂ -terminal alanine found in ₂ .

"Low concentration of IL-2" refers to a dose capable of stimulating and activating LAK cells in a range that does not temporarily increase the activity of the virus in the human body or cause side effects such as cytokine leak syndrome. Preferably, when the amount of IL-2 secreted from LAK cells to the human body per day is about 500,000 IU or less.

As described above, immunotherapy with LAK cells injects high concentrations of IL-2 along with LAK cells to maintain sustained survival of the LAK cells. Such high concentrations of IL-2 increase secondary latency cells of the HIV virus. In addition, systemic administration of IL-2 causes side effects of leakage of intravascular fluid into the extravascular space (capillary leak syndrome) (Rosenstein M. et al., Immunology, 137, 1735-1742 (1986); Ohkubo); C. et al., Cancer Res., 51, 1561-1563 (1991); Edwards, MJ et al., Cancer Res., 52, 3425-3431 (1992); Damle NK et al., J. Immunol., 142, 2660-2669 (1989). Capillary leakage syndromes caused by IL-2 administration include edema formation, hypotension and renal dysfunction. In the case of administering IL-2 to humans for the purpose of tumor treatment, the dosage can be tolerated only up to 10% of the optimal dose as derived from animal studies. However, even at doses commonly used, lethargy, nausea and vomiting, diarrhea, fluid retention, hypotension and organ dysfunction are observed, even leading to death.

"Cancerous cells", unlike normal cells, are cells that autonomously proliferate and destroy surrounding tissues and develop invasive, for example, pancreatic cancer cells, lung cancer cells, colon cancer cells, liver cancer cells, breast cancer cells, and gynecological cancer cells. And prostate cancer cells, bladder cancer cells, skin cancer cells, soft tissue cancer cells, and the like. Preferably, it includes malignant melanoma cells, lung cancer cells, kidney cancer cells, colon cancer cells and rectal cancer cells and the like.

"Virus-infected cells" are normal cells that have been transformed by the virus and have the following characteristics: they grow easily in vitro, grow much faster than normal cells, and cause many changes on the cell surface (increasing ions' ability to pass, binding toxin hormones) Loss of capacity, new antigen production, etc.) and chromosomal aberration occurs, and an antiviral agent called interferon is produced. In particular, viral infection herein means infection by a Human Immunodeficiency Virus (HIV). HIV is the pathogen of the Acquired Immunodeficiency Syndrome (hereinafter referred to as AIDS). The main target of HIV is helper T cells among T cells that regulate immune function. When secondary T cells are infected with HIV and cause necrosis, the immune function of the human body is impaired, causing an immunodeficiency state, which causes fatal infections and malignant tumors.

"Selectively killing" means the ability of the I-LAK cells according to the present invention to lyse and remove the cells by showing cytotoxicity to cancer cells or cells infected with the virus without showing cytotoxicity to normal cells. The present inventors examined the cytotoxicity of I-LAK cells against various cells such as HIV-infected peripheral blood mononuclear cells, K562, CEM, ACH.2, H9, H9 / HTLV-III _MN and the like. It was found that the cells selectively killed HIV-infected cells and malignant cells without damaging the cells (Example 5, Fig. 3).

I-LAK cells according to the invention have the following characteristics:

1) Since LAK cells are IL-2 dependent, their survival may be maintained in the environment in which IL-2 is provided (Grimm EA et al., J Exp Med 158 (4), 1356-61 (1983)), but IL- I-LAK cells into which two genes are introduced can maintain cell survival even if no separate IL-2 is provided. This is because the present inventors cultured LAK cells and I-LAK cells without a stimulant such as IL-2 for 60 days, and LAK cells showed a survival rate of 0% at 60 days of culture, whereas I-LAK cells had a 60-day survival rate. It was confirmed that the excellent 90% (Example 4, Figure 2).

2) IL-2 secreted from I-LAK cells reduces the total amount of cells in the incubation period in humans by stimulating HIV-infected cells out of incubation period to escape the incubation period (Chun TW et al., J. Exp. Med). 188 (1), 83-91 (1998. 12)).

3) Since I-LAK cells secrete IL-2 at low concentrations, it is possible to prevent the transient virus activation that occurs when LAK cells and high concentrations of IL-2 are administered to HIV infected persons according to conventional methods (Ramratnam B. et al., Nat. Med. 6 (1), 82-5 (2000. 09)).

4) IL-2 is secreted continuously in I-LAK cells, which may increase the immunity of HIV infected. In this regard, IL-2 is known to increase the number of cells chronically important for immunity of CD4 + T cells to HIV infected individuals (Davey RT Jr. et al., JAMA, 284 (2), 92074 (2000)).

In another embodiment of the present invention, the present invention provides a method of (a) bleeding peripheral blood from a mammal and removing red blood cells from the peripheral blood to obtain a lymphocyte-containing leukocyte fraction, and (b) a culture medium containing IL-2. And cultured in (C) the resulting LAK cells, (c) transfected with the recombinant vector containing the IL-2 gene and cultured under appropriate medium and conditions to differentiate the cells, (d) culture It provides a method for producing I-LAK cells introduced IL-2 gene, characterized in that it comprises the step of recovering the cells having the IL-2 secretion ability in the cells.

More specifically, peripheral blood is first collected from mammals and centrifuged, Ficoll-paque is added, and then centrifuged again to obtain peripheral blood mononuclear cells.

Peripheral blood mononuclear cells are then suspended in LAK activation medium with IL-2 added to induce production of LAK cells.

The LAK cells obtained above were transfected with a retroviral vector, preferably a multiple gene expression recombinant retroviral vector LNC / IL-2 / IRES / TK, in which IL-2 and a thymidine kinase gene of herpes simplex virus were simultaneously expressed. Let's do it. The transfected cells were cultured in a medium containing G418 (neomycin analog) to obtain G418 resistant cells, and the culture supernatant thereof was assayed for detection of IL-2 secretion, for example IL-2 using ELISA. The cells excellent in secretion capacity are selectively recovered to obtain I-LAK cells of the present invention.

Recombinant vectors exemplified in the present invention are LNC / IL-2 / IRES / TK. The synthetic retroviral vectors are known and can be obtained from Dr. Yeon-Soo Kim of KAIST (Korea Advanced Institute of Science and Technology).

The recombinant vector LNC / IL-2 / IRES / TK is a thymidine kinase (TK) that enables treatment of cells transfected with a gene expressing IL-2 and a recombinant vector gene with an antiviral agent through a CMV promoter. Genes are included. TK gene expression significantly increases sensitivity to, for example, Ganciclovir (GCV). Preferably, the LTR promoter / neomycin marker / CMV promoter, IL-2 gene, internal ribosomal transduction site gene and tyrosine kinase gene are sequentially linked to act (FIG. 1).

The term "vector" refers to a DNA preparation containing a DNA sequence operably linked to a suitable regulatory sequence capable of expressing the DNA in a suitable host. When transformed into a suitable host, the vector can replicate and function independently of the host genome or in some cases integrate into the genome itself. In the present invention, the host is a eukaryotic cell.

A wide variety of expression vectors can be used to express the DNA sequences according to the invention. Suitable expression vectors for eukaryotic hosts include, for example, SV40, bovine papilloma virus, retrovirus, Adenovirus, herpes simplex virus, poxvirus, lentivirus. , Adeno-associated virus and cytomegalovirus.

Preferred expression vectors herein are retroviral vectors. Retroviruses are a group of viruses in which the RNA genome is converted into DNA in infected cells and inserted into the chromosome of a host cell. Retrovirus vectors based on MoMLV (Moloney Murine Leukemia Virus) are the most widely used. Retroviral vectors have the advantages of being easily manipulated in vitro, efficient gene delivery, and genes that can be stably inserted into the chromosome of a host cell and continuously delivered to progeny cells. The LNCX retroviral vector exemplified herein has the neo ^r marker gene expressed by the 5'LTR promoter and the CMV immediate-early promoter of human cytomegalovirus as an internal promoter. Therefore, there is a characteristic that the gene introduced from the outside can be expressed with high efficiency.

The expression "expression control sequence" refers to a DNA sequence essential for the expression of a coding sequence operably linked in a particular host organism. Such regulatory sequences include promoters for performing transcription, any operator sequence for regulating such transcription, sequences encoding suitable mRNA ribosomal binding sites, and sequences that control termination of transcription and translation. For example, regulatory sequences of eukaryotic cells include promoters, polyadenylation signals, enhancers, and the like.

Nucleic acids are "operably linked" when placed in a functional relationship with other nucleic acid sequences. This may be genes and regulatory sequence (s) linked in such a way as to enable gene expression when appropriate molecules (eg, transcriptional activating proteins) bind to regulatory sequence (s). For example, DNA for a pre-sequence or secretion leader is operatively linked to DNA for a polypeptide when expressed as a shear protein that participates in the secretion of the polypeptide; A promoter or enhancer is operably linked to a coding sequence when it affects the transcription of the sequence; Ribosome binding sites are operably linked to coding sequences when they affect transcription of the sequence; Ribosome binding sites are operably linked to coding sequences when placed to facilitate translation. In general, "operably linked" means that the linked DNA sequences are in contact, and in the case of a secretory leader, are in contact and present within the reading frame. However, enhancers do not need to touch. Linking of these sequences is performed by ligation (linking) at convenient restriction enzyme sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers according to conventional methods are used.

Recombinant vectors can be introduced into cells using conventional transformation methods such as DEAE-dextran, calcium phosphate, electroporation. Techniques for transforming host cells and methods for expressing foreign DNA sequences cloned therein are well known in the art (Maniatis et al., Molecula Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory (1982); Sambrook et al., Supra; Gene Expression Technology, Method in Enzymology, Genetics and Molecular Biology, Methods in Enzymology, Guthrie & Fink (eds.), Academic Press, San Diego, Calif. (1991); Hitzeman et al., J Biol Chem., 255, 12073-12080 (1980); US Patent No. 4,935,349). Preferably, the recombinant vector according to the present invention transfects host cells by calcium phosphate mixed precipitation.

In another aspect of the present invention, the present invention provides a pharmaceutical composition for treating cancer or viral infection disease comprising an amount of said I-LAK cells sufficient to elicit an immune response.

The pharmaceutical composition may comprise a pharmaceutically acceptable carrier. Many suitable carriers or diluents can be used in the present invention, particularly saline, buffered saline, and saline mixed with nonspecific serum albumin. Pharmaceutical compositions may contain water, saline, glycerol, ethanol and the like or emulsions, wetting agents or pH, in addition to adjuvant, buffers, antioxidants, carbohydrates such as glucose, sucrose or dextrin, and chelating agents such as EDTA. Regulating substances can be included. The pharmaceutical composition may comprise an adjuvant to enhance the immune response. Examples of the adjuvant include aluminum hydroxide (alum), thr-MDP, nor-MDP, MPT-PE and the like.

The pharmaceutical composition can be administered parenterally, intramuscularly, subcutaneously, intradermal, intraperitoneally, intranasally or intravenously or via a different route suitable for the tumor or infection and the condition of the patient being treated. Relatively non-invasive methods are preferred, with subcutaneous routes preferred, as low levels of inflammation or induration may occur for several days after administration.

The pharmaceutical composition of the present invention is administered in an amount sufficient to have an effect that can cause an immune response to the individual. In the pharmaceutical compositions of the present invention, the dosage is typically about 10 ⁵ to 10 ¹¹ I-LAK cells. Preferably about 10 ⁶ to 10 ¹⁰ cells are used, more preferably about 1 x 10 ⁷ to 2 x 10 ⁹ cells. However, the amount may vary depending on the extent to which protection is required, the age or weight of the individual, the formulation of the immunotherapeutic composition, the method of administration and the like.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following examples are illustrative of the present invention, and the content of the present invention is not limited by the examples.

 (Example)

Example  One: LAK  Preparation of cells

 Peripheral blood was collected by venipuncture from a healthy HIV-negative donor (HIV antibody-negative). Percol-paque was added to the blood and then centrifuged to obtain peripheral blood mononuclear cells. Peripheral blood mononuclear cells were cultured in RPMI-1640 supplemented with 15% fetal bovine serum (FBS), penicillin, streptomycin and L-glutamine. The culture was treated with rIL-2 (aldesleukin, Chiron BV Amsterdam, Netherland) 1000 IU / ml for 7 days to induce the production of LAK cells.

Example  2: recombinant expression vector LNC Of / IL-2 / IRES / TK Construction

 In order to introduce IL-2 gene into LAK cells of Example 1, two effects genes IL-1 and thymidine kinase gene of Herpes simplex virus (HSV tk gene) Multiple gene expression retroviral vector LNC / IL-2 / IRES / TK, in which the gene is expressed simultaneously, was constructed.

The recombinant vector LNC / IL-2 / IRES / TK according to the present invention inserts the HSV TK gene into the retroviral vector LNCX, connects the internal ribosomal introduction site (IRES), and sequentially introduces the IL-2 gene back into the preparation. It was constructed by (FIG. 1).

Introduction of the HSV TK gene was synthesized by PCR using DNA fragments from which the promoter and poly (A) signal sites were removed from pTK3 (BRL, Gaithersburg, MD) encoding the gene. The DNA fragment was made into phosphorylated blunt ends at both ends by T4 DNA polymerase and T4 polynucleotide kinase and inserted into retroviral vector LNCX digested with restriction enzyme Hpa I. . LNCX retroviral vectors are genes introduced from the outside as viral vectors in which neo ^r marker genes are expressed by the 5 'LTR promoter and the early-early promoter (CMV promoter) of human cytomegalovirus is included as an internal promoter. Highly efficient expression of can be expected. The LNC / TK clones in which the HSV tk gene of the recombinant retroviral vector was expressed from the CMV promoter were identified and selected by restriction enzyme digestion and nucleotide sequencing. Upstream of the HSV-tk gene, the plasmid pCITE- is introduced to introduce a cap-independent translation enhancer sequence containing an internal ribosome entry site (IRES) of the encephalomyoca rditis virus (EMC). About 600 bp EcoRI-NcoI fragment was extracted from 1 (Novagen, WI, USA) and subcloned into the recombinant vector LNC / TK (LNC / IRES / TK). To introduce the IL-2 gene back into this plasmid, approximately 700 bp of the HindIII-BamH1 IL-2 DNA fragment (Invivogen, USA) was subcloned between the CMV promoter and the IRES sequence to retroviral vector LNC / IL-2 / IRES / TK. Was constructed.

Example  3: Preparation of Retrovirus Packaging Cell Lines

* LNC / IRES / TK or LNC / IL-2 / IRES / TK plasmids for the production of retroviral packaging cell lines producing retroviruses encoding the HSV tk gene and / or the IL-2 gene. DNA was transfected into an amphotropic packaging cell line PA317 (ATCC, USA) by calcium phosphate coprecipitation. After 48 hours, G418 was replaced with medium containing 600 g / ml and incubated for 10-14 days until G418-resistant colonies were produced. Each resulting colony was isolated and expanded to mass culture. In order to screen virus-producing cell lines with high titers, serial dilutions of the virus-producing PA317 / LNC / IRES / TK cells and PA317 / LNC / IL-2 / IRES / TK cells were performed. x 10 ⁵ NIH / 3T3 cells were cultured in 60-mm diameter petri dishes and then infected for 4 hours by inoculation of 1 ml in the presence of 8 g / ml polybrene after 24 hours. After adding 4 ml of fresh culture and incubating for 48 hours, the cells were treated with trypsin and then inoculated into a 100-mm diameter culture dish and selected cultured for 10 to 14 days in a culture containing 400 g / ml G418. The experiment was repeated three times independently, from which the virus titers of each cell line were calculated. As a result, the highest titer cell line was obtained.

Example  4: I- LAK  Preparation of cells

 On the 10th day after the start of the production of the LAK serotypes, the LAK cells were transformed with the LNCX / IL-2 / IRES / TK gene under the control of the CMV promoter into I-LAK cells using a retroviral vector. 48 hours after retroviral vector was added to LAK cells, the cells were washed and cultured in RPMI-1640 medium containing G418 (neomycin analog) to differentiate the cells associated with neoR gene. G418-resistant cells were obtained and their culture supernatants were measured by ELISA to investigate IL-2 secretion capacity. RT-PCR confirmed the insertion of the IL-2 secretion gene in I-LAK cells.

Example  5: LAK  And I- LAK  Measurement of Viability of Cells

 LAK cells and I-LAK cells were cultured for 60 days in a medium containing no cell stimulant. Cell viability was measured by trypan blue exclusion.

As a result, the viability of LAK cells at 10 days of culture was 98%, 63% at 20 days, 40% at 30 days, 3% at 40 days, 2% at 50 days and 0% at 60 days. I-LAK cells showed higher viability compared to LAK cells, showing 97% on day 10, 97% on day 20, 95% on day 30, 95% on day 40, 90% on day 50 and 90% on day 60 (FIG. 2).

Example  6: LAK  Cells and I- LAK  Cytotoxicity measurement of various target cells of cells

Cytotoxicity of I-LAK cells and LAK cells against various target cells was measured by a ⁵¹ chromium release assay.

As target cells, normal peripheral blood mononuclear cells, HTLV-III _MN -infected peripheral blood mononuclear cells, K562, CEM, ACH.2, H9 and H9 / HTLV-III _MN were used. K562, a target cell line of LAK cells, was used as a positive control in LAK cytotoxicity assay. CEM cell lines were used to compare the level of LAK cell activity of CD4 + human cell line ACH.2. ACH.2 is a T cell subclone A3.01 potentially infected with HIV-1 and infected with HIV _LAV . Cell line originated from CEM. The human T cell line was isolated from a 4 year old Caucasian female, acute lymphoblastic lymphoma. H9 and H9 / HTLV-III _MN are single cell clones of specific HUT 78 cell line origin. ACH.2, H9 and H9 / HTLV-III _MN were donated by the NIH AIDS Research and Reference Regent Program. All the cells were cultured in RPMI-1640 with 1% FBS, penicillin, streptomycin and L-glutamine.

The target cells 310 ⁶ were incubated in a water bath at 37 ° C. for 10 hours using 0.5 ml of medium containing 150 μl of ⁵¹ Cr. ⁵¹ Cr-labeled cells were washed four times with fresh medium and 50 μl of cells were plated in 96-well plates to 10 ⁴ cells per well. LAK cells and I-LAK cells were added to the plates in ratios of 20: 1, 1: 1 and 1:20. The effector and target cells were incubated for 8 hours at 37 ° C., 5% CO ₂ incubator, and then 50 μl of the supernatant was taken from each well to perform isotope counting. All analyzes were repeated three times, and the percentage of lysis was calculated by the following equation.

The maximum release was determined by incubating 50 μl of 0.5% Trypton X-100 with 50 μl of target cells. Spontaneous release was determined by culturing 50 μl of cell culture and 50 μl of target cells. Spontaneous release was always less than 20% of maximum release (Antonelli P et al., Clin. Immunol. Immunopath. 19, 161-169 (1981)). Various parameters between LAK cells and I-LAK cells were compared using the Student's T-test. When p value was 0.05 or less, it was judged to be statistically significant.

As a result, both LAK and I-LAK cells showed strong cytotoxicity against the cells of tumor cell lines, K562, CEM, ACH.2, H9, H9 / HTLV-III _MN . This is generally because LAK cells are involved in the killing of malignant cells and cells infected with viruses. Malignant cells and HIV infected cells were all killed and removed selectively, while normal cells were not damaged. Although there was no statistical difference, the specific lysis rate of ACH.2 cells tended to be higher than that of CEM cells, and the specific lysis rate of H9 / HTLV-III _MN cells was higher than that of H9 cells. On the other hand, there was no significant difference in the selective cytotoxicity of LAK and I-LAK cells to HIV infected cells (FIG. 3).

Example 7 Comparison of HIV Production in Mixed Culture of LAK Cells or I- LAK Cells with HIV-Infected Cells

To prepare HIV-infected peripheral blood mononuclear cells, HIV virus strain stocks were prepared and used to infect peripheral blood cells. HIV virus strain (HTLV-III _MN ) stock was prepared by recovering culture supernatant when virus production rate of infected H9 cells was the highest. All HIV stocks were determined titers by determining TCID ₅₀ endpoint 7 days after cell infection. HIV infection of peripheral blood cells was induced by incubating HIV at mois 1.0 at 37 ° C. for 12 hours. Control cells were obtained by culturing uninfected H9 cell culture supernatants. Infected peripheral blood cells were centrifuged, washed and resuspended in fresh medium. Half of the culture supernatant was replaced with fresh medium every three days. Cell viability was measured by trypan blue exclusion.

Mixed cultures of LAK cells or I-LAK cells with HIV-infected peripheral blood cells and the amount of virus liberated in the cell culture medium were separated every three days using the HIV-1 p24 antigen-capturing EIA kit (Organon Teknika, Boxtel, Netherlands). Quantification by For each experiment, a standard curve was prepared and the amount of p24 was calculated in consideration of the number of dilutions of the sample, the standard curve, and the OD value.

As a result of the quantification of the p24 antigen of HIV collected from the culture supernatant of H9 / HTLV-III _MN cell line, it was found that the most viral replication occurred at 6 days of culture. In the case of mixed culture of H9 / HTLV-III _MN and LAK cells, the p24 antigen value decreased until 15 days of culture and then increased again from day 18 of culture. The increase in the p24 antigen value (the rebound in viral replication) is thought to be due to the reduced viability of LAK cells from 2 to 3 weeks in the absence of IL-2 stimulant. In the mixed culture of H9 / HTLV-III _MN and I-LAK cells, the p24 antigen value continued to decrease by about 21 days. Viral replication increased by 6 days in culture with peripheral blood cells infected with H9 / HTLV-III _MN . In the mixed culture of HIV-infected peripheral blood cells and LAK cells or I-LAK cells, p24 antigen levels were significantly decreased from 9 to 21 days of culture. This demonstrates in vitro that the ability of LAK cells and I-LAK cells to kill HIV-infected cells is similar (FIG. 4).

 Lymphocine-activated killer cells I-LAK according to the present invention secrete low concentrations of interleukin-2 and have the activity of selectively killing cancer cells or virus-infected cells without damaging normal cells, thereby treating cancer and treating viral infections. Very useful for

In the above, in order to help the description and understanding of the present invention in more detail through specific examples, various changes are possible within the scope of the present invention as will be apparent to those skilled in the art. Accordingly, the description and examples should not be considered as limiting the claims of the invention.

1 is a schematic diagram of the recombinant vector LNC / IL-2 / IRES / TK according to the present invention (LNC: LTR promoter / neomycin marker / CMV promoter, IRES: internal ribosomal introduction site, TK: thymidine kinase, MSV: mouse Sarcoma virus, MLV: murine leukemia virus, SD: splicing provider, SA: splicing recipient).

Figure 2 is a graph comparing the cell viability of LAK cells and I-LAK cells.

3 is a graph showing cytotoxicity of various target cells of LAK cells and I-LAK cells.

Figure 4 is a graph showing the results of quantifying HIV production by EIA method when mixed culture of LAK cells or I-LAK cells and HIV-infected cells.

Claims (3)

An anticancer or antiviral pharmaceutical composition comprising an anticancer or antiviral lymphokine active killer cell (I-LAK cells) transfected with a recombinant vector operably linked to an interleukin-2 gene in an amount sufficient to elicit an immune response. . The method of claim 1, wherein the amount sufficient to cause the immune response is Pharmaceutical composition wherein 10 ⁵ to 10 ¹¹ I-LAK cells. The pharmaceutical composition according to claim 1 or 2, wherein said antivirality is against HIV virus.