MXPA99003758A - Targeted cytotoxic cells - Google Patents
Targeted cytotoxic cellsInfo
- Publication number
- MXPA99003758A MXPA99003758A MXPA/A/1999/003758A MX9903758A MXPA99003758A MX PA99003758 A MXPA99003758 A MX PA99003758A MX 9903758 A MX9903758 A MX 9903758A MX PA99003758 A MXPA99003758 A MX PA99003758A
- Authority
- MX
- Mexico
- Prior art keywords
- cell
- cells
- immunotoxin
- antigen
- antibody
- Prior art date
Links
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Abstract
Disclosed are mammalian cells that express and secrete immunotoxins directed against tumors, HIV antigens and other diseased cells. Preferred cells include lymphocytes and neurons.
Description
TARGET CITOTOXIC CELLS
BACKGROUND OF THE INVENTION
1. Field of the Invention The present invention relates generally to the fields of microbiology and immunotherapy. More particularly, it relates to antigen-specific cytotoxic cells that provide antibody-mediated and cell-mediated immunity.
2. Description of the Prior Art Immunities mediated by antibody and cell use different mechanisms to defend a host against tumor growth and viral infections. Immunity of antibody particularly functions in the neutralization of pathogens through antibody-dependent cell annihilation (Stites). et al., 1994) T cell-mediated immunity utilizes the functions of the two subsets of T cell helper T cells that mediate their effect through the secretion of lymphokines to activate other effector cells, and cytotoxic T cells that annihilate target cells through activation mediated by the receptor of apoptosis (Berke, 1994, Young and Liu, 1988) Increasing numbers of preferentially expressed antigens on the cell surface have been identified
for tumor cells, such as oncoproteins and cell differentiation antigens, and these may provide potential markers for the targeted destruction of target cells in cancer therapy (Urban and Shreiber, 1992; Pastan and FitzGerald, 1991; Boon et al. , 1994). Two fundamental aspects for tumor immunotherapy have been explored: the target aimed at the antibody of toxic and cytolytic activity to tumor cells (Vitetta et al., 1987; Waldmann, 1991; Dohlsten et al., 1994), and the increase in immune responses. Cells to tumors (Pérez et al., 1985; Gross et al., 1989; Goverman et al., 1990; Rosenberg, 1991; Eshhar et al., 1993; Hwu et al., 1993). Although antibody-directed immunotherapy has been shown to effectively kill tumors in vitro as well as in vivo, a major obstacle to this successful use in cancer therapy is the limited accessibility of antibodies or antibody conjugate to solid tumors (Jain, 1989; Shockey et al., 1992; Reithmuller and Johnson, 1992). In these cases, due to limited access to cancer cells, immunotherapy directed to the antibody can not completely destroy the target tumor. For this type of immunotherapy to be successful, successful means must be found to deliver antibodies or antibody conjugates into a tumor. Considerable efforts have also been made to stimulate or modify T lymphocytes through the transduction of cytokine genes. , or antibody / T cell receptors to increase the
cellular antitumor activities (Rosenberg et al., 1988, Rosenberg et al., 1990, Kawakami et al., 1994). But a major limitation in the use of this type of adoptive cell immunotherapy is the difficulty in obtaining specific cytotoxic nuclides (Rosenberg et al., 1988 , Rosenberg et al., 1990, Kawakami et al., 1994) Unless specific cytotoxic lymphocytes are obtained, specific cytotoxicity can occur and result in unacceptable side effects to the patient. The use of naturally occurring toxins to treat different types of cancer Viral infections currently have a very limited range Plants and bacteria produce defense toxins that are among the most potent toxins capable of killing mammalian cells, that is, one or some molecules are sufficient to kill a mammalian cell (Yamaizumi and others, 1978, Chen and others 1995) For example, plants produce nema, abpna, gelonina and sapopnas The bacterial toxins include exotocin Pseudomonas A (PEA) and diphtheria toxin. Toxin molecules, such as PEA, kill a cell by blocking protein synthesis through the inactivation of elongation factor-2 (EF-2) in the cytosol (graze) and FitzGerald 1992) Unfortunately, the therapeutic use of these and other plant or bacterial toxins is very limited due to the fact that the cytotoxic effects of the toxins are not limited to specific cell types such as tumor cells or virally infected cells and consequently, the
Nonspecific cell and tissue death that can result in unacceptable side effects or risk to the patient In addition, these toxins currently must be injected systemically and are cleared from the blood stream and the body very quickly. For therapeutic uses, it may be desirable toxins remain in the system for an extended period so that they can "sweep" target cells that were not removed or killed by other treatments, for example, in the treatment of metastatic tumor cells It is evident from vast research efforts to fight cancer and viral diseases since new methods to treat these diseases of death are necessary In particular, a great benefit can be obtained from a treatment method that improves the specific cytotoxic character towards target cells, especially cells where access is limited , such as cells within solid tumors and that allows toxins to remain present in the system as "sweepers", and simultaneously reduce the dangerous side effects and risks for the patient
COMPENDIUM OF THE INVENTION
The present invention seeks to overcome these limitations by providing a surprising improvement in the field of immunotoxic therapy since the subject's own cells or other
mammalian cells can be used to produce and secrete an immunotoxin, and those cells can then be infused back into the subject to act as a therapeutic or preventive agent in the treatment of the target disease. The invention provides several improvements with respect to the therapies of conventional immunotoxin, wherein the immunotoxin is synthetically prepared and then injected into a subject. For example, in the practice of the present invention, immunotoxins can be produced in a cell culture and conveniently purified from the culture medium simply by filtration, centrifugation and chromatography, in contrast to conventional means to produce immunotoxins such as chemical entanglement of toxins to antibody molecules Another difficulty with conventional immunotoxins is that they rapidly disappear from the circulation in a subject, requiring multiple administrations, and it being difficult to maintain therapeutic levels in the blood without incurring significant side effects. In contrast, in the practice of the present invention, the cells that produce the immunotoxin are viable in the body of a subject and produce immunotoxins for periods of weeks to months from a single infusion In a certain broad aspect, the present invention can be described as a mammalian cell such as a cytotoxic T lymphocyte, a neuron or another mammalian cell that expresses and secretes an immunotoxin. The immunotoxin is preferably
encoded by a vector comprising a nucleic acid sequence encoding a leader sequence, the expression of which directs the secretion of the immunotoxin out of the cell into a secretion vesicle before the expressed toxin can damage the cell of origin or host Once the immunotoxin is secreted, the cytotoxic effect is mainly limited to those cells that express the antigen that is recognized by the antibody component of the immunotoxin, and the cell of origin, which could normally express a high level of the antigen , remains viable and continues the expression of immunotoxin A cytotoxic T cell (annihilation cell) is known in the art as an immune system cell involved in the immunocellular response that has the ability to recognize a foreign antigen and kill the cell by presenting that antigen An immunotoxin is known in the art as a function or conjugation of an antibody component and a toxin component such that the antibody or domain component is capable of specifically recognizing and binding an antigen presented on a target cell, for example, and thus directing the toxin to that particular cell type. As used herein, "expresses and secretes" indicates that the cytotoxic T cell or other mammalian cell of the invention contains a segment of genetic material that encodes an immunotoxin, and that the genetic material is translated into a polypeptide immunotoxin product through the translation (ie pbosome) mechanisms of
the host cell, and further that the immunotox is transported outside the host cell secreted into the extracellular environment. In the practice of the invention, the genetic material encoding the immunotoxin may comprise a DNA vector or an RNA vector, or the material can be incorporated into genomic material of the host cell The immunotoxin of the present invention is understood to comprise an antibody component and a toxin component, illustrated herein as co-expressed from a single promoter. However, an immunotoxin of the invention can also be expressed as two or more mRNA messages expressed from separate promoters, whose translation products are then assembled into the cell, or during or after the secretion procedure. The antibody domain of the immunotoxin may comprise an anti-tumor antibody domain in certain embodiments, or may comprise an antiviral antibody domain in certain embodiments or may comprise an anti-T cell helper antibody domain or a specific subgroup of T-helper cells in further embodiments of the invention. For example, the immunotoxin may be immuno-reactive with a HER2 antigen, a Lyn-1 antigen, a growth factor or a hormone receptor or any other known antigen that is over expressed in a tumor cell Antiviral immunotoxins can be immunoreacted with an HIV antigen such as an antigen gp120 or an influenza virus, a herpes virus a
Epstem-Barr virus or any known viral antigen In an alternative modality, anti-T helper cell immunotoxins can immuno react with a specific subgroup of T helper cells which are involved in an autoimmune disease Preferred toxins that will be used in practice of the invention include, but are not limited to Pseudomonas exotocins, such as exotocin A Pseudomonas, nema A, diphtheria toxin, or a portion thereof with cytotoxic and transport activities, abin, gelonin, sapopan or any other toxin for which the coding gene is available. In a certain aspect, the invention may comprise a mammalian cell such as a cytotoxic T-cell or neuron that expresses and secretes an immunotoxin, wherein the cell is dispersed in a pharmaceutically acceptable vehicle solution. This solution may be suitable for injection, either intramuscularly or intravenously, or it may be adequate for inhalation or other means of administration known in the art The phrase "pharmaceutically acceptable" refers to compositions that do not produce a similar allergic or undesired reaction when administered to an animal or a human being, and which may include, but are not limits to any and all solvents, media, isotonic agents and the like The use of such media and agents for pharmaceutically active substances is well known in the Except art since any conventional media or agent is incompatible with the active ingredient, its use in the compositions
The additional therapeutic ingredients can be incorporated into the compositions. In another broad aspect, the invention can be described as an animal cell, a lymphocyte, a cytotoxic lymphocyte or a neuron transfected with a vector, so that the vector comprises a nucleic acid sequence operably linked to a promoter, and the nucleic acid sequence encodes one or more pbosomal binding sites and a fusion protein comprising a leader sequence of an immunotoxin, and all the genetic signals necessary for the expression of a gene eukaryotic such as a pohadenylation site In this embodiment of the invention, the expressed immunotoxin of the vector is trans-located to the endoplasmic reticulum of the hnfocyte directed by the leader sequence. Thus, it is understood that the leader sequence is expressed before the expression of the toxin of the immunotoxin and it is preferable that the first coding region is expressed from the promoter The vector can be any suitable expression vector known in the art, in which the leader and immunotoxin genes are inserted, and can be a plasmid, a viral vector such as an adenoviral, retroviral, or adenoviral vector associated with a cosmid or other type of vector known in the art. In addition, the vector can comprise a natural DNA or RNA molecule that is associated with a liposome or lipid complex, or the vector can be introduced into a cell through receptor-mediated gene transfer through electroporation and through
of other mechanical, electrical or chemical means known in the art Immunotoxin expressed in the vector described above can immuno-react with an antigen associated with the tumor, such as HER2 or Lym-1 antigens or with a viral associated antigen, such as an antigen. HIV antigen, or even an HIV antigen gp120 or with an antigen associated with a certain subgroup of T helper cells In certain embodiments, the host animal cell, or lymphocyte may comprise a pharmacological composition, when the cell is dispersed in a pharmaceutically acceptable carrier, for example, as described above The present invention can be described in a broad aspect as a method for killing a tumor cell, the method comprising contacting the tumor cell with an immunotoxin secreted from an animal cell, such as a cytotoxic T cell or a neuron, where the antibody portion of the immunotoxin recognizes an antigen of the tumor cell Preferably, the tumor cell on expresses HER-2 or Lym-1, or another antigen associated with tumor and the tumor cell can be a B cell lymphoma, a breast tumor, ovarian, gastric cell or brain, or another tumor cell known to overexpress a surface antigen compared to a normal cell. In preferred embodiments, the tumor cell is an animal subject, and most preferably a human being, or even a human patient with cancer, and the Animal cell is administered to the subject in a pharmaceutical composition The invention can also be described
in a broad aspect as a method for inhibiting the tumor cell or metastatic tumor cell growth in a subject comprising administering a cytotoxic T cell and antitumor cell expression immunotoxin to a subject. The immunotoxins described herein may also be expressed and secreted through a neuron or other brain cell Fusion proteins of this embodiment can be described as comprising a peptide signal sequence, an antibody domain and a cytotoxin component, wherein the antibody domain is immuno -reactive with an antigen associated with a brain tumor cell, and wherein the production and secretion of an immunotoxin or brain cell does not adversely affect the host brain cell as described above. This modality is contemplated as being particularly effective since neurons that express autologous immunotoxin can be directly implant in a brain after surgical removal of a tumor, for example for long-term expression of an immunotoxin against residual tumor cells or metastases. In a broad aspect the present invention can also be described as a method for killing a virally infected cell that comprises contacting the cell with an immunotoxin expressed from a cytotoxic T cell, or another mammalian cell wherein the antibody portion of the immunotoxin recognizes an antigen expressed by the cell
virally infected In the practice of this embodiment, a cytotoxic T cell can be administered in a pharmaceutical solution to an animal or a human being having a virally infected cell. In certain broad aspects, the present invention can be described as a method of inhibiting a HIV infection in a subject, which comprises administering a pharmaceutical solution to the subject, wherein the solution comprises a cytotoxic T cell that expresses and secretes an anti-HIV immunotoxin. In a broad aspect, the present invention can also be described as a method for annihilate a specific species of T helper cells, wherein the specific species of T helper cells are involved in an autoimmune disease, and the antibody portion of the immunotoxin recognizes an antigen specifically expressed by the particular group of T helper cells., certain subgroups of T helper cells can be over-reactive in such conditions as rheumatoid arthritis or systemic lupus ephematose, for example The invention offers advantages over therapies that suppress the entire immune system, since only the specific cells involved in the condition can be the target and the rest of the immune system is left intact. Another additional advantage in the case of rheumatoid arthritis for example, is that the cells that produce immunotoxin can be injected directly to the site of the condition, such as in a joint. this modality the cytotoxic hnfocitos or cells that produce
immunotoxin can be administered in a pharmaceutical composition to an animal, most preferably to a human, who has an autoimmune condition where a specific subgroup of T helper cells is involved. The present invention can also be described in a broad aspect as a method for producing a recombinant immunotoxin, wherein the method comprises the steps of obtaining a mammalian cell transfected with an expression vector encoding a leader sequence and a mmunotoxin After introduction of the expression vector into the mammalian cell, or after transfection of the cell with the expression vector, an immunotoxin is expressed and secreted from the cell into the culture medium. In the practice of this method, the leader immunotoxin construct can be expressed from a variety of promoters known in the art, including the CMV promoter or even an inducible promoter when desired The leader can direct the expressed immunotoxin towards the endoplasmic reticulum or secretion vesicle of the cell, when the cell is cultured under effective conditions to express the immunotoxin. The method for producing an immunotoxin can further comprise the step of isolating the immunotoxin from the medium of This method offers many advantages over the chemical synthesis of immunotoxins, or even expression in a bacterial cell such as E Coli. For example large amounts of immunotoxins can be produced in a continuous cell culture and easily isolated from the cell.
Culture medium through a variety of methods known in the art In addition, the expression of a mammalian gene in a bacterium usually results in insoluble inclusion bodies in which the proteins are not correctly bent and can be inactive. invention offers the advantage that active immunotoxins can be easily isolated from the culture medium through standard techniques. These isolated immunotoxins can be packaged for commercial sale, and can be reused for in vitro applications, or research, or for infusion therapies in where whole cells are not desirable
BRIEF DESCRIPTION OF THE DRAWINGS
The following drawings are part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention can be better understood by referring to one or more of these drawings in combination with the detailed description of the specific embodiments presented herein. Schematic representation of the construction of the anti HER2 / toxin expression vector The expression vector pCmv-sFv23e-PE40, contains an ant? -HER2 sFv23e gene (Batra et al. 1992 Kasprzyk et al. 1992 Bird et al. 1988 Marasco et al. 1993 ) with a leader signal sequence fused to the sequence
PE40 (domains II and III) of PEA (ATCC), all under the control of the CMV promoter In the recombinant retroviral promiscuous vector, LNCX-sFve23 / PE40, the sFve23 / PE40 gene is activated by the internal CMV promoter and the resistance gene a neomycin (neo) is activated by LTR Figure 2 ADP-pbosylation activity of secreted sFv23e-PE40 fusion toxins The culture medium of MOLT-sFv23e-PE40 and MOLT-Control was harvested and subjected to ADP-pbosylation analysis as described they exist previously (Colher and Kandel, 1971, Chen et al., 1995). Purified PEA proteins were denatured through urea as previously described (Col I le r and Kandel, 1971) and then used in ADP-pbosylation assays. Mean values of the submitted samples are calculated from duplicate determinations after subtracting the background level. The filled columns represent the PEA activity at the indicated concentrations. The open columns indicate Control medium Moit-4, culture medium of MOLT-sFv23e-PE40 (sFv23e-PE40-1) MOLT-sFv23e-PE40 (sFv23e-PE40-1), culture medium of MOLT-sFv23e-PE40 concentrated through of an Amicon filter (sFv23e-PE40-2) Figure 3A Tumor cells (SKOV-3, N-87 over-expressing HER2, and control cell lines (MCF-7 and NIH3T3) expressing low or undetectable levels of HER2 that were planted in 96-well plates (1x105 / ml) After 24 hours of incubation, 0 3 ml of Molt-sFv23e-PE40 cells (clones 1 and 2) or Molt control cells (5x105 / ml) were added to each well of the plate, and co-cultures were continued for 62 hours. Dead cells in these co-cultures they were then classified through tppan blue staining (Chen et al., 1995), and selective cell annihilation percentages are shown after subtracting the percentage of dead cells (3%) in co-cultures of tumor cells with control cells Molt Figure 3B The percentage inhibition of cytotoxicity when the indicated concentrations of the 23e antibody of origin were added to co-cultures of Molt sFv23e-PE40 (clone 1) and the SKOV3 tumor cells Figure 4A Tumor cells (N87) (5x106) were injected subcutaneously to the flanks of athymic / Nu mice and developed for 16 hours The Molt-sFv23e-PE40 and Molt control cells were washed with PBS and resuspended in the medium e culture after counting cell numbers Mice with xenograft grafts were randomized into two groups to the treatment group (7 mice) were administered Molt-sFv23e-PE40 (1x10 in 0 2 ml), and the control group (5 mice) were given Molt control cells (1x107 in 0 2 ml) on day 1 followed by weekly injections for six weeks. Tumor diameters were measured per gauge every three to five days and the tumor volume was calculated at through the formula tumor volume = (width) 2 x long? tud / 2 (Osborne et al., 1985)
The mean tumor volumes of the treatment group (filled diamonds connected through a solid line) and the control group (solid diamonds connected through a dotted line) are shown. The differences of growth curves between the treatment group and the control were statistically significant (P = 0009), as analyzed by a Manova test Figure 4B The survival of the treatment groups (solid diamond) and the control groups (solid box) is shown as described in Figure 4A Figure 5 Schematic diagram of the expression vector of ant? -gp120 / tox? Na To generate a specific annihilation cell in HIV-1, a human neutralizing monoclonal antibody (f105) was used that recognizes the CD4 binding site of HIV- 1 gp120 expressed on the surface of cells infected with HIV-1 The gene (PE40) encodes domain II (for transubicación through the double membrane layer) and domain III (pair adenosine biphosphate (ADP) -r? bos? lac? on EF-2) of PEA was fused to the K-chain gene of F105 The resulting bicistronic vector (pCMV-Fabl 05-PE40) contains an Fd chain (VH + CH?), An internal pbosomal entry site (IRES) and the chimeric gene? G-PE40 predicted by a leader signal sequence under the control of the cytomegalovirus (CMV) promoter In the recombinant retroviral vector LNCX-Fab 105-PE40, the chimeric Fac105 / PE40 gene is expressed from the internal CMV promoter and the NEO gene is
expressed from LTR The construction was identified through restriction enzyme digestion and confirmed through DNA sequence analysis Figure 6A The antigen binding activity of the Fab105-PE40 fusion proteins detected through ELISA The activities Positive binding has HIV-1 gp120 were detected in the medium of Jurkat-Fab105-PE40, while no significant binding activity was observed in the control medium Jurkat The solid columns are the culture medium of Jurkat-Fab105-PE40 (Fab105-PE40), Jurkat control culture medium (Jurkat) Open columns positive HIV-1 gp120 binding activities through indicated concentrations of purified F105 antibody (0 8 to 0 1 μg / ml) Figure 6B ADP activity -Posylation (DPB) of Fab105-PE40 The culture medium of Jurkat-Fab105 cells and Jurkat control was subjected to ADP-pbosylation assays The solid columns are Jurkat-Fab105-PE4 culture medium 0 (Fab105-PE40-1), Jurkat-Fab105-PE40 culture medium concentrated through Amicon filtration (Fab105-PE40-2) Jurkat control culture medium (Jurkat) Open columns ADP-pbosylation activity of Fab105-PE40 measured through indicated concentrations of PEA purified (5 to 40 ng per reaction) Figure 7 Selective cytoxicity to HIV-1 infected cells m vitro through transduced lymphocytes Jurkat cells of origin were infected with an HIV-1 strain of
laboratory (IIIB) and two primary patient isolates (INME and TPO), and reverse transcpptase (RT) activities were measured every three to four days until the activity reached leveling. The Jurkat cells infected with HIV-1 were then placed in upper chambers of 12-well Costar-Transwell filter tissue culture plates, and Jurkat-Fab105-PE40 and Jurkat control cells were placed in the background chambers at 1 1 ratios (Jurkat-HIV infected cells). Fab105-PE40 or Jurkat control cells) (open columns), and 10 (solid columns) Viable cell numbers of cells infected with HIV-1 in the upper chambers were counted at 72 hours after co-culture, and Percentages of cell annihilation were expressed as percentage of viable cell numbers of infected cells co-cultured with Jurkat-Fab105-PE40 as compared to number of viable cells of infected cells c o-cultured with Jurkat control Figure 7B Selective cell annihilation of transduced LAK cells Human LAK cells were generated by incubating peripheral blood lymphocytes in a culture medium supplemented with r? L-2 and PHA for 48 hours LAK cells were transduced with the Fab105-PE40 gene through co-cultures with the transfected packaging cell line (PA317) for 24 hours followed by expansion for several days. Transduced LAK cells were co-cultured with HIV-infected cells from a laboratory strain HIV-1 (IIIB) and two strains of
primary patient (INME and TPO) for 72 hours, and the percentages of cell annihilation were presented as previously described Open column ratio 1 1 of HIV-infected cells LAK cells transduced or transduced by mock Solid column ratio 1 10 of infected cells HIV LAK cells transduced or transduced by mock Figure 7C Kinetics of inhibition of HIV-1 infection Jurkat cells of origin infected with the patient's primary HIV-1 isolate (WEAU) were co-cultured with Jurkat-Fab105-PE40 cells or Jurkat control cells at ratios of 1 1, 1 5 and 1 10 The RT activities in the co-cultures were checked every three to four days after co-cultivation
DESCRIPTION OF THE ILLUSTRATIVE MODALITIES
The present invention provides a new class of cytotoxic cells that combine the specificity of antibodies, extreme potency of toxins and the effector cell properties of hnfocytes such as lodging and tissue penetration, thus offering the advantages of both antibody directed immunotherapy and cell-mediated cytotoxic lymphocytes not only recognize the target, but also produce potent molecules to kill the target cells in an independent form of major histocompatibility (MHC). Furthermore, the cells described are not adversely affected by the
produced immunotoxins Surprisingly, cytotoxic hnfocytes not only remain viable but do not appear to be damaged in any way. The present invention can also be described as cytotoxic cells that are capable of expressing and secreting an immunotoxin, which comprises a leader signal sequence, a domain of immunologically active antibody that recognizes an antigen specific to the target cell, and a cytotoxin The immunotoxins produced by the cells of the invention described have a high specific character towards their target cells and tissues so that no non-specific toxic interaction is detected with non-target cells or tissues. The cells are capable of producing the immunotoxin molecules for a sustained period of up to three months or more. A further aspect of the immunotoxin secreting cells of the present invention is that the cells can be designed with a marker of selection negative for reasons of patient safety Incorporating a marker gene into the recombinant vector that is used to transduce the cells, such as two tandem copies of the simple herpes virus thymidine kinase (HSV-tk) gene that provides negative selection in the presence of ganciclovir (Ishibashi et al., 1993), the transduced cells can be selectively destroyed if necessary. could allow the doctor to selectively destroy the immunotoxin secretion cells in case of adverse side effects
This new site of cytotoxic cells, which are capable of producing and secreting novel target toxin proteins, was designed based on the knowledge of protein trafficking and the mechanism of cell annihilation of toxins. The toxin molecules, such as exotoxin A Pseudomonas (PEA), block the cellular protein synthesis and in this way annihilate the cell inactivating the elongation factor-2 (EF-2) in cytosol (Vitetta et al., 1987, Waldmann, 1991, Dohlsten et al., 1994, Yamaizumi et al., 1978, Hwang et al., 1987, Siegall et al., 1989, Pastan et al., 1992), indicating that toxin molecules have been located in the cytosol in order to kill a cell. The present invention exploits this property, genetically modifying a cell to produce and secrete target toxins using a leader signal sequence (Walter and Lingappa, 1986). Subsequently the target toxins, recently synthesized, are transub They are located in the lumen of the endoplasmic reticulum (ER) cotranslationally and then secreted out of the cells. The cells expressing toxin must remain viable, since the interaction of the fusion toxins synthesized with the EF-2 in the cytosol is blocked by the double layer of membrane ER membrane and secretion vesicles Selectively secreted target toxins bind and destroy target cells after being internalized and released into the cytosol (Vitetta et al. 1987, Waldmann 1991 Dohsten et al., 1994, Yamaizumi et al. , 1978 Hwang and others 1987 Siegall and others 1989, Pastan and others 1992)
However, the secreted toxins are not able to kill the toxin-expressing cells, due to the lack of target antigens on the surface of the cells. The genetically modified cells have a potent and selective cytotoxicity to the target cells indicating that this aspect may have a broad explanation for the treatment of viral infection, cancer and autoimmune diseases This aspect of the present invention can be used in therapeutic applications to treat a viral infection, in particular an HIV-1 infection wherein the transduced lymphocytes can lodge and secrete anti -VIH / toxins in lymphoid tissues and organs, the major deposits of HIV-1 infection (Pantaleo et al., 1993, Embretson et al. 1993). Thus, relatively high levels of locally produced target toxins may be more effective in neutralizing cell-free vipones and destroy HIV-infected cells, compar With the systemic administration of antibody / toxin proteins (Chaudhary et al. 1988) The aspect of the present invention can also be used to selectively destroy other targets, such as tumors, since the numbers of antigen increase selectively expressed on the surface of tumor cells have been identified by antibodies (Pastan and FitzGerald, 1992 Walter and Lmgappa, 1986 Waldmann, 1991) For example breast cancer cells can be recognized and destroyed through transduced cytotoxic cells secreting
Target Toxins Certain embodiment of the present invention may be useful in the treatment of certain diseases and autoimmune reactions, such as rheumatoid arthritis. This type of disease is usually aggravated by a specific subgroup of over-reactive T helper cells. The present invention provides a method to specifically inhibit the dangerous T cells by directing an immunotoxin only to those cells This provides an advantage over the therapies of autoimmune diseases where the complete immune system is inhibited, since the target immunotoxin will not affect other elements of the immune response mechanism of the subject, including other subgroups of T helper cells and which does not cross-react with the antibody In the practice of this embodiment, the dangerous species of T helper cells can be isolated from a subject, and used to produce an antibody that recognizes an antigen specific to the subgroup of T helper cells through standard antibody production techniques known in the art. This antibody can then be used as described herein to produce an immunotoxin vector and the vector can then be used to transduce a lymphocyte, for example that could secrete the target immunotoxin to the T helper cells. The transduced cells can also be infused directly to the site of inflammation, such as a joint, in order to have a better target. towards the site of the
Disease In summary, this new class of cytotoxic cells specific to antigen and fusion proteins with the aspects of cell-mediated antibody-directed immunotherapy may have broad applications for the treatment of viral infection, cancer and autoimmune diseases and reactions As shown in the present, transduced cytotoxic cells have been found to have a potent selective cytoxicity for target cancer cells in vitro as well as in vitro. In addition to combining the specificity of antibodies to the potency of toxin molecules and the effector cell properties of hnfocytes , these cells have been shown to have specific antitumor activity in vivo without obvious non-specific toxicity One aspect of the invention is the use of tumor infiltration hnfocytes (TlLs) or other lymphocytes, since TlLs can rapidly prophylact in vitro and recirculate in and be located in tumor sites after Unes of reinfusion (Perez et al., 1985, Gross et al., 1989, Goverman et al., 1990, Rosenberg, 1991, Eshhar et al., 1993, Hwu et al., 1993, Rosenberg et al., 1988, Rosenberg et al., 1990 , Kawakam et al., 1994, Bolhuis et al., 1991, Topahan et al., 1989, Barth et al., 1990). The tumor lodging characteristic of transduced TlLs also allows them to function not only as a vehicle to deliver the antibody. toxins to tumor tissues but also as a producer of target toxins within tumor tissues. It is especially evident that tumor micrometastasis is almost
impossible to detect, it can be selectively destroyed through these target cytotoxic cells when they are reinfused to patients, acting as an immune monitor
Immunotoxins Immunotoxin technology is quite well advanced and is known to those skilled in the field of antibody screening Immunotoxins are agents that have an antibody component bound to another agent, particularly a cytotoxic or otherwise anticellular agent having the ability to annihilate or reduce cell growth or cell division Illustrative anti-cell agents include chemotherapeutic agents and radioisotopes as well as cytotoxins Examples of chemotherapeutic agents are hormones such as antimetabohto steroids such as cytokine rabinoside, fluoracil methotrexate or aminopeptepne, anthracycline mitomycin C, vinca alkaloids, demecolcine, etoposide, nitramycin or antitumor alkylating agents such as chlorambucil or melphalan Preferred immunotoxins usually include a toxin derived from fungal plants, or bacteria such as a protein A chain toxin na of ribosome inactivation, a-sarcin aspergillin restpctocma a pbonuclease such as placental pbonuclease angiogenin, diphtheria toxin or exotoxin of Pseudomonas sapopna gelonina or abpna to mention only
Some examples It is an important element of the present invention that the toxin is co-expressed genetically with the antibody in the lymphocyte or other mammalian cell. Therefore, the preferred toxins that will be used are those for which the coding genes are known. and that they can be inserted into an expression vector through standard molecular biology techniques. The use of any of these toxins in the practice described herein could be encompassed by the spirit and scope of the present invention. For example, now a chain A of biologically active nema has now been cloned and expressed (O'Hare et al., 1987, Lamb et al., 1985, Halling et al., 1985), so that nema A, or smaller or otherwise vanishing peptides which nevertheless exhibit an appropriate toxin activity can be used in the practice of the present invention. In addition, the fact that the nema A chain has now been cloned allows the application of site-directed mutagenesis, whereby A chain-derived peptides can be readily prepared and screened and additional useful portions can be obtained for use in conjunction with the present invention
Generation of Monoclonal Antibody Means for preparing and characterizing antibodies are well known in the art (see for example Antibodies A Laboratory Manual Cold Spring Harbor Laboratory, 1988, incorporated herein by
reference) Methods for generating monoclonal antibodies (MAbs) generally begin along the same lines as those for preparing polyclonal antibodies In summary, a polyclonal antibody is prepared by immunizing an animal with an immunogenic composition, such as a specific species of helper cells T, according to the present invention (either with or without prior immunotolerance, depending on the composition of antigen and protocol being used) and collecting antisera from that immunized animal. A wide variety of animal species can be used for production of antisera Typically, the animal used for the production of antisera is a rabbit, a mouse, a rat, a hamster, a guinea pig, or a goat. Due to the relatively large blood volume of rabbits, the rabbit is a preferred selection for the production of polyclonal antibodies As is well known in the art, a This composition may vary in its immunogenicity It is usually necessary, therefore, to strengthen the host immune system, and can be achieved by coupling a peptide or polypeptide immunogen or a carrier. Illustrative and preferred carriers are keyhole hemocyanin. (KLH) and bovine serum albumin (BSA) Other albumins such as ovalbumin mouse serum albumin or rabbit serum serum albumin can also be used as carriers The means to conjugate a pohpeptide or a
carrier proteins are well known in the art and include glutaraldehyde, m-maleidobenzoyl-N-hydroxysuccinimide ester, carbodnmide and bis-diazotized benzisma. As is well known in the art, the immunogenicity of a particular immunogen composition can be improved through the use of non-specific stimulators of the immune response, known as adjuvants Illustrative and preferred adjuvants include Freund's complete adjuvant (a non-specific stimulant of the immune response containing annihilated Mycobacterium tuberculosis), incomplete Freund's adjuvants and aluminum hydroxide adjuvant The amount or The immunogen composition used in the production of polyclonal antibodies varies depending on the nature of the immunogen as well as the animal used for immunization. A variety of routes can be used to administer the immunogen (subcutaneous, intramuscular, mdermic, intravenous and mpepeptoneal). The polyclonal antibody can be verified by sampling the blood of the immunized animal at a few points after immunization. A second boost of reinforcement can also be provided. The reinforcement and titration process is repeated until an adequate titration is achieved. When a desired level is obtained of immunogenicity, the unimmunized animal can be bled and the serum isolated and stored and / or the animal can be used to generate MAbs
MAbs can be readily prepared through the use of well-known techniques, such as those illustrated in US Patent 4,196,265, incorporated herein by reference. Typically, this technique involves immunizing a suitable animal with a selected immunogen composition, for example, a purified or partially purified T cell surface protein, polypeptide or peptide or any other desired antigenic composition The immunization composition is administered in an effective manner to stimulate the cells that produce antibodies Rodents, such as mice and rats, are preferred animals without However, the use of rabbit or lamb cells is also possible. The use of rats may provide certain advantages (Goding, 1986, p. 60-61), but mice are preferred., the BALB / c mouse being very preferred since this is the most commonly used and generally provides a higher percentage of stable fusions After immunization, somatic cells with the potential to produce antibodies, specifically B cells (B cells), are selected for use in the MAb generation protocol. These cells can be obtained from spleens that were extracted by biopsy, tonsils or lymph nodes, or from a peripheral blood sample. Basal cells and peripheral blood cells are preferred. , the former because they are a rich source of antibody production cells that is in the division plasmablast stage and the latter because the
Peripheral blood is easily accessible Generally, a panel of animals will be immunized and the spleen of the animal with the highest antibody titre will be removed and the baso lymphocytes obtained by homogenization of the baso with a syringe Typically, a baso of a mouse immunized contains approximately 5 X 107 to 2 X 108 of lymphocytes. The B lymphocytes of antibody production of the immunized animal are then fused with cells of an immortal myeloma cell, generally one of the same species with which the animal was immunized. Myeloma cells suitable for use in hybridization production processes of hibpdoma are preferably non-antibody producing, have high fusion efficiency, and enzyme deficiencies that make them then unable to grow in certain selective media that support the growth of desired fused cells (hibpdomas) Any number of cells of Myeloma can be used, as is known to those skilled in the art (Goding, p. 65-66, 1986, Campell, p. 75-83, 1984) For example, when the immunized animal is a mouse, one can use P3-X63 / Ag8, X63-Ag8 653 NS1 / 1 Ag 4 1 Sp210-Ag14, FO, NSO / U, MPC-11, MPC11-X45-GTG 1 7 and S194 / 5XX0 Bul, for rats one can use R210 RCY3, Y3- Ag 1 2 3 IR983F and 4B210, and U-266, GM1500-GRG2, LICR-LON-Hmy2 and UC729-6 all are useful in relation to human cell fusions
A preferred murine myeloma cell is the melanoma cell line NS-1 (also referred to as P3-NS-1-Ag4-1), which is readily available from the NIGMS Human Genetic Mutant Cell Repositiory by requesting the line deposit number GM3573 cell phone Another line of mouse melanoma cell that can be used is the non-producing cell line SP2 / 0 of mouse murine myeloma resistant to 8-azaguanine. Methods for generating basal or lymph node cell hybrids of antibody production and myeloma cells usually comprise mixing somatic cells with myeloma cells in a ratio of 2: 1, although the ratio can vary from about 20: 1 to about 2: 1, respectively in the presence of an agent or agents (chemical or electrical) that promote the fusion of cell membranes. Fusion methods using Sendai viruses have been described by Kohler and Milstein (1975; 1976) and those using polyethylene glycol (PEG), such as 37% (v / v) PEG, by Gefter et al. (1977). The use of electrically induced fusion methods is also appropriate (Goding pp. 71-74, 1986). Fusion procedures usually produce viable hybrids at low frequencies, from about 1 X 106 to 1 X 10"8 However, this does not pose a problem since it is viable, the fused hybrids are differentiated from unfused origin cells (particularly unfused myeloma cells that could normally continue to divide indefinitely)
culturing in a selective medium. The selective medium is generally one containing an agent that blocks the de novo synthesis of nucleotides in the tissue culture medium. Illustrative and preferred agents are aminopetepne, methotrexate and azasepna. Aminopeptepna and methotrexate block de novo synthesis of both pupils and pipmidines, whereas azasepna blocks only pupil synthesis. When aminopetepne or methotrexate is used, the medium is supplemented with hypoxanthine and thymidine as a source of nucleotides (HAT medium). When azasepna is used, the medium is supplemented with hypoxanthine The preferred screening medium is HAT Only cells capable of operating wild nucleotide trajectories are able to survive in a HAT medium. Myeloma cells are defective in key enzymes of the wild-type pathway, for example hypoxantma-phosphobosyl transferase ( HPRT), and can not survive B cells can operate this tray ectoria, but they have a limited life span in culture and generally die in approximately two weeks., the only cells that can survive in the selective medium are those hybrids formed from myeloma and B cells. This culture provides a population of hibpdomas from which specific hibpdomas are selected. Typically, the selection of hibpdomas is done by culturing the cells through individual clone dilution in microtiter plates, followed by the individual clonal supernatant test (after
approximately two to three weeks) for the desired activity. The analysis should be sensitive, simple and rapid, such as radiommunoassays, enzyme immunoassays, cytotoxicity assays, plaque assays, dot immunostaining assays, and the like. The selected hibpdomas then separately diluted and cloned to individual antibody production cell lines, said clones can then be propagated indefinitely to provide MAbs. Cell lines can be exploited for the production of MAb in two basic forms. A sample of the hibpdoma can be injected (usually at the peptoneal cavity) to a histocompatible animal of the type that was used to provide the somatic and myeloma cells for the original fusion The injected animal develops tumors that secrete the specific monoclonal antibody produced by the fused cell hybrid Fluids from the animal's body, such as serum or ascites fluid, after can be trapped to provide MAbs in a high concentration Individual cell lines can also be cultured in vitro, when MAbs are naturally secreted into the culture medium from which they can be easily obtained in high concentrations MAbs produced either by any medium can be further purified, if desired using centrifugation filtration and various chromatographic methods such as HPLC or affinity chromatography
Immunoassays Immunoassays that will be used in the practice of the present invention include, but are not limited to those described in U.S. Patent No. 4,367,110 (double monoclonal antibody sandwich assay) and U.S. Patent No. 4,452,901 (Western dye). Other Assays include immunopresorption of labeled gandos and immunocytochemistry, both mv in vivo Immunoassays, in their simplest and most direct sense, are binding assays. Certain preferred immunoassays are the various types of enzyme-linked immunosorbent assays (ELISAs) and other immunoassays. solid supports known in the art Very preferred are ELISAs as described by Doellgast et al. (1993 1994) and by US Patent No. 4,668,621. Immunohistochemical detection using tissue sections and radioimmunoassays (RIAs) are also particularly useful. easily appreciated that detection is not limited to such technique s, and that Western staining, spot staining, FACS analysis, and the like can also be used. In an illustrative ELISA assay, the antibodies of the invention, ie those that will be used in the production of an immunotoxin are immobilized on a surface selected exhibiting protein affinity, such as a cavity in a polystyrene microtiter plate. Next, a biological sample suspected of containing the target antigen which itself may be linked to a detectable label.
is added to the cavities After joining and washing to remove non-specifically bound immunocomplexes, the amount of bound antigens can be determined. Alternatively, the first aggregate component that remains bound within the primary immune complexes can be detected through a second binding ligand having binding affinity for the primary antibody In these cases, the second binding ligand can be linked to a detectable label The second binding ligand is itself an antibody, which can thus be referred to as a "secondary" antibody Immunoprimary complexes are contacted with the secondary binding ligand, labeled, or antibody, under effective conditions and for a sufficient period to allow the formation of secondary immune complexes. Secondary immune complexes are generally then washed to remove any antibody or secondary labels labeled, not specifically bound, and the remaining label in secondary immune complexes is then detected. This type of ELISA analysis is a simple "sandwich ELISA" analysis. Other methods include the detection of primary immune complexes through a two-step approach. A second binding ligand such as an antibody, which has binding affinity for the primary antibody is used to form secondary immune complexes, as described above After washing
the immune secondary complexes are contacted with a third binding ligand or antibody having binding affinity for the second antibody, again under effective conditions and for a sufficient time to allow the formation of immune complexes (tertiary immune complexes). The third ligand or antibody is bound to a detectable label, allowing the detection of the tertiary immune complexes thus formed. The system can provide signal amplification if desired. In another illustrative ELISA assay, samples suspected of containing the target antigens are immobilized on the cavity surface and then contacted with antibodies or immunotoxins of the invention After binding and washing to remove non-specifically bound immunocomplexes, bound antigens are detected. When the initial antibodies are linked to a detectable label, immunocomplexes can be detected directly Once again, immunocomplexes can be detected using a second antibody that has binding affinity for the first immunotoxin antibodies, the second antibody being bound to a detectable label. Another ELISA analysis in which the proteins or peptides are immobilized involves the use of antibody competition in detection In this ELISA assay, labeled antibodies are added to the cavities and binding is allowed and detected through their label The amount of target antigens in a
Unknown sample is then determined by mixing the sample with the labeled antibodies before or during incubation with coated cavities. The presence of the target antigens in the sample acts to reduce the amount of immunotoxin antibodies available for attachment to the cavity and thus reduces the final signal Regardless of the format used, ELISA assays have certain common aspects, such as coating, incubation or binding, and washing to remove non-specifically bound species, and the detection of bound immunocomplexes. These are described as follows. a plate with either antigen or antibody, the cavities of the plate will usually be incubated with a solution of the antigen or antibody, either overnight or for a specific period of time. The cavities of the plate will then be washed to remove the material incompletely. absorbed Any remaining available surface of the cav They are then "coated" with a non-specific protein that is antigenically neutral with respect to the test antisera. These include bovine serum albumin (BSA), casein and milk powder solutions. The coating allows the blocking of non-adsorbing sites. specificities on the immobilization surface and thus reduces the background caused by the non-specific binding of antisera on the surface In ELISA assays, it is probably customary
using a secondary or tertiary detection means instead of a direct procedure In this way, after the binding of a protein or antibody to the cavity, the coating with non-reactive material reduces the background, and the washing removes the unbound material, the immobilization surface is contacted with the control antigen and / or biological sample that will be tested under conditions in a conducted form to allow for the formation of immune complex (antigen / antibody). The detection of the immune complex then requires a ligand or antibody from the immunocomplex. labeled secondary binding, or a secondary binding ligand or antibody together with a third binding ligand or labeled tertiary antibody "Under conditions in a conducting manner to allow the formation of immunocomplex (antigen / antibody)" may include diluting the antigens and antibodies with solutions such as BSA, bovine gamma globulin (BGG) and saline regulated in its pH c on phosphate (PBS) / Tween These aggregate agents also tend to aid in the reduction of non-specific background. The 'proper' conditions also represent that the incubation is at a temperature and for a sufficient time to allow an effective binding. The incubation steps typically they are approximately 1 to 2 to 4 hours, at temperatures preferably of the order of 25 to 27 ° C, or it can be overnight at about 43 ° C etc. After all the incubation steps in a test of
ELISA, the contact surface is washed in order to remove the material that did not form in complex A preferred washing procedure includes washing with a solution such as PBS / Tween, or borate pH regulator After the formation of specific immunocomplexes between the test sample and the originally bound material, and subsequent washing, the occurrence of even minimal amounts of immune complexes can be determined. To provide detection means, the second or third antibody will have an associated label to allow detection. Preferably, this will be an enzyme that will generate color development after incubation with an appropriate chromogenic substrate. Thus, for example, if it is desired to contact and incubate the first or second immunocomplex with a urease, glucose oxidase alkaline phosphatase or antibody conjugated with peroxidase of hydrogen for a time and under conditions that favor the development of the f Additional immunocomplete ormation (eg incubation for 2 hours at room temperature in a solution containing PBS such as PBS-Tween) After incubation with the labeled antibody and after washing to remove unbound material, the amount of label is quantitated, for example, by incubation with a chromogenic substrate such as urea and bromocresol purple or 2 2-azene-d? - (3-et? l-bemzot? azol? na-6-sulfon? co [ ABTS] and H2O2 in the case of peroxidase as the label of
enzymes Then, the quantification is achieved by measuring the degree of color generation, for example, using a spectrophotometer with visible spectra
Promoters and Enhancers The promoters and enhancers that control the transcription of genes that encode protein in mammalian cells are composed of multiple genetic elements. The cellular machinery is able to gather and integrate the regulatory information transported by each element, allowing different genes to develop different transcpptional regulation complex patterns The term promoter will be used here to refer to a group of transcourse control modules that are assembled around the initiation site for RNA polymerase II. Much with respect to how the promoters are organized is derived from analysis of several viral promoters, including those for HSV thymidine kinase (tk) and SV40 early transcription units These studies, augmented by more recent work, have shown that the promoters are composed of discrete functional modules, each consisting of approximately 7-20 bp of DNA, and containing one or more recognition sites for trans-transcriptional activator proteins. At least one module in each promoter functions to place the start site for RNA synthesis. The best known example of this is the TATA box but in some
promoters lack of a TATA box such as the promoter for the mammalian terminal deoxycytidyl transferase gene and the promoter for the final genes SV 40 a discrete element covering the start site by itself helps to set the initiation site The elements Additional promoter regulates the frequency of transcourse initiation Typically, these are located in the 30-110 bp region upstream of the initiation site, although it has recently been shown that a number of promoters contain functional elements downstream of the initiation site as well. between the elements is flexible, so that the promoter function is conserved when the elements are inverted or moved relative to each other. In the tk promoter, the separation between the elements can be increased to 50 bp before the activity starts to decline Depending on the promoter, it seems that the individual elements can work either cooperative or independently to activate the transcription The breeders were originally detected as genetic elements that increased the transcription of a promoter located at a distant position on the same DNA molecule. This ability to act over a great distance had a small precedent in classical studies of regulation Procapotic transcpplication Subsequent work showed that DNA regions with enhancer activity are organized much
as the promoters that is, they are composed of many individual elements, each of which binds to one or more transcpptional proteins. The basic distinction between breeders and promoters is operational. An enhancer region as a whole should be able to stimulate transcription to a distance, this need not be true for a promoter region or its component elements. On the other hand, a promoter must have one or more elements that direct the initiation of RNA synthesis at a particular site and in a particular orientation, while the breeders lack these specifications Also in the operational distinction, the breeders and promoters are very similar entities They have the same general function of activating the transcription in the cells They are usually overlapping and contiguous, they almost always seem to have a very similar modular organization Tomadas Together, these considerations suggest that the breeders and Promoters are homologous entities and that the transcourse activating proteins bound to these sequences can interact with the cellular transcourse machinery in essentially the same way. Below is a list of viral promoters / cellular promoters / enhancers and inducible promoters / enhancers that can be used in combination with the construction of immunotoxins In addition any combination of promoter / enhancer (AS PER the Eukaryotic Promoter Data Base EPDB) can also be used to direct the expression of the
Immunotoxin fusion gene in a gene therapy protocol TABLE 1
TABLE 2
USE OF PROMOTERS AND IMPROVERS
It is understood in the art that to give a coding sequence under the control of a promoter, the 5 'end of the transcription initiation site of the transcriptional reading frame of the protein is placed between about 1 and about 50 nucleotides "downstream" of (ie, 3 'of) the selected promoter. In addition, when eukaryotic expression is contemplated, it will also typically be desirable to incorporate into the transcriptional unit including the co-transporter protein, an appropriate pohadenylation site (e.g., 5'-AATAAA-3 ') if one was not contained in the Original cloned segment Typically, the poly A addition site is placed from about 30 to 2000 nucleotides "downstream" of the protein termination site at a position before the termination of the transcript
Recombinant Vectors
For use in mammalian cells, the control functions in the expression vectors are usually provided by the viral material. For example, the commonly used promoters are derived from cytomegalovirus (CMV), polyoma, Adenovirus 2, and very frequently virus. Siam 40 (SV40) Early and subsequent SV40 virus promoters are particularly useful since both are easily obtained from viruses as a fragment that also contains the replication SV40 viral origin (Fiers et al, 1978) SV40 fragments can also be used smaller or larger, provided that a sequence of approximately 250 bp is included extending from the Hmd lll site to the Bg1 I site located in the viral origin of replication. Also, if possible, and generally desirable, sequences are used. promoter or control normally associated with the desired gene sequence, provided that said control sequences are compatible with host cell systems The origin of replication can be provided either through construction of the vector to include an exogenous origin, such as can be derived from CMV, SV40 or another viral source (eg Polynesis Adeno VsV, BPV), or can be provided through the mechanism of chromosomal rephcacion host cell If the vector is integrated into the host cell chromosome, the latter is usually sufficient
Expression Vector Construction
The DNA fragment encoding amino acids 253 to 613 of PEA (ATCC) was fused in frame to the K gene of the human monoclonal antibody anti? -VIH-1 gp120, F105 The bicistronic expression vector containing the Fd-IRES -? - F105 was previously constructed (Chen et al, 1994), and was used to construct the expression vector pCMV-Fabl 05-PE40 (FIG 5) The resulting bicistronic expression vector, pCMV-Fabl 05-PE40, contains the FdF105 gene / internal ribosome entry site (IRES) sequence / k105-PE40 fusion gene under the control of a CMV promoter This expression vector was identified with restriction enzyme digestions, and confirmed by sequence analysis DNA In this expression vector, only one mRNA was transcribed under the control of the CMV promoter, and the two gene products were independently translated from a single mRNA. The translation of the first Fd105 gene is cap-dependent, and the second gene is fusion? -PE40 was tra ducido under the control of IRES in the cap-independent manner A more detailed description of the vector construction is presented below
Construction of Bicistronic Expression Vector for Fab105 Using IRES
The co-expression of the heavy and light chains of the
Fab105 fragments were achieved from a Fab105 expression vector using two independent CMV promoters (Chen et al, 1994) The co-expression vector of the Fab105 expression cassette with two identical CMV promoter sequences was further modified to include a sequence of internal ribosome entry site (IRES) The IRES derived from EMCV has been used for efficient co-expression of two or more genes in retroviral vectors and other vectors In this type of expression vector, a single mRNA is transcribed under control of an upstream promoter, and the two gene products are independently translated from an individual mRNA The translation of the first gene is cap-dependent, and the second is translated under the control of IRES in an independent form of cap The expression vector Fab105 bicistronic using an internal ribosome entry site sequence (IRES) was constructed from the expression vector using two Identical CMV promoters as follows the IRES sequence of the encephalomyocarditis virus (EMCV) was amplified by PCR ™ from a plasmid pCITE-2a (Novogen, Madison, Wl) using the primers (5'-TTTGCTAGCGGTATTATCATCGTG-3 (SEQ ID NO 1) with an additional Nhel cloning site, 5'-TTTGCGGCCG CGAATTAATTCCGGTTA-3 '(SEQ ID NO 2) with an additional Notl site) The IRES DNA fragment, approximately 515 bp was purified from an agarose gel, and then it was cut with Nhe \ / Not \
To induce a single Nhel cloning site to the bicistronic expression vector with IRES, a light chain (lambda) fragment of a human neutralizing monoclonal antibody 2.1H against the CD4 binding site of HIV-1 gp120 was amplified by PCR ™ to from the hybridoma cDNA and sequenced (Bagley et al., Molecular Immunology, 31: 1149-1160, 1994) The 2.1H light chain fragment was then cut with NhellXbal and gel purified. The 2.1H-cut light chain of NhellXbal and the Notl / Nhel cut and the IRES DNA fragments were cloned to NotllXbal cut pCMV-Fab105 through a three-piece ligature The resulting construct, pCMV-F105-IRES-2 1H , which contains Fd of F105, IRES, and the 2.1H light chain sequence under the control of the cytomegalovirus (CMV) promoter, was identified and confirmed through DNA sequencing The plasmid pCMV-Fd105-IRES-2.1 H it was then digested with Nhel and Xoal, and fragments of the DNA vector were recovered from an agarose gel. The K-chain DNA fragments of F105 were amplified by PCR ™ from pCMV-Fab105 using the primers 5 -GGTTGCTAGCATGGAAACCCCAGCGCAG-3 '(SEQ ID NO 3) 5'-AAAATCTAGATTAACACTCTCCCCTGTTGAA-3' (SEQ ID NO'4) Amplified chain fragments, approximately 760 bp, were digested with Nhel and Xbal, and purified through agarose gels pCMV-Fd 105-IRES-2 1H cut with NhellXbal and the DNA -K fragments were then ligated together and transformed to a host E. coli The expression vector
resulting bicistronic, pCMV-Fabl 05-1, which contains the Fd / IRES genes / under the control of CMV promoter, was identified through enzyme digestions, and used for further study
Construction of Bicistronic Expression Vector for Fab105-PE40 Fusion Protein
The PEA gene obtained from the American-type culture collection (ATCC) contains three functional domains: Domain I, cell recognition, Domain II, translocation domain (amino acid residues (aa) 253 to 404), and Domain III, catalytic domain. (aa from 405 to 613) (Pastan and FitzGerald, Science 25 1173-1177, 1992) Bg / ll-EcoRl fragments containing the sequence encoding amino acids 253 to 613 of PEA were cut from plasmid pJH8 (ATCC), and cloned to vector pSP72 (Stratagene) (pSP-PE40) To incorporate a NotI site into the fragment, an initiator corresponding to amino acid residues 253 to 258 of PEA with an additional NotI site (5 -TTGCGGCCGCGAAAGGCGGCA GCCTGGCCGCG-3 (SEQ ID NO 5) and an inverse primer corresponding to amino acids 330 to 324 (5'-GCGGATCGCTT CGCCCAGGT-3 '(SEQ ID NO 6) were used to amplify amino acids 253 to 330, and amplified DNAs were then cut with NotUSal I fragment. Salt l / Xbal-containing DNA the aa sequence 308 to 613 of PEA was cut and purified from the pSP-PE40 vector The NotUSal I fragment from aa 253/308 and the
UXbal salt DNA fragment encoding aa 308/613 was then cloned to the NotUXbal sites of pCMV-sFv23e-S through a three-part ligation. The NotUXbal DNA fragment containing the PE40 sequence (Domain II and III) was cut from the pCMV-sFv23e / PE40, and the K-chain gene was amplified by PCR ™ from the pCMV-Fabl 05-I vector to incorporate the Nhel / Notl sites. The resulting bicistronic expression vector for Fab105-PE40 was constructed through a ligation of three pieces of the cutting chain? /? / oíl / cut? / ofl / Xoal / cut-PE40 /? /? el / X < bal / cut-pCMV-Fab105-I The vector was identified through restriction enzyme digestion and confirmed by DNA sequencing
RadioetiQuetation and Immunoprecipitation
For transient expression, COS cells were transfected with
μg of pCMV-Fabl 05-PE40 of plasmid DNA or the pRc / CMV vector using Lipofectin (Chen and Compans, 1991) and, were radiolabeled with 200 μCi of 35S-c? Steine for several times after 60 to 72 hours transfection The transduced Jurkat cells (5 x 10 °) were radiolabelled with 200 μCi of 35S-cysteine for several times. Culture medium and cell lysates were immunoprecipitated with a mixture of anti-PEA and anti-human IgG. through electrophoresis in SDS-poliacplamide gels under reduction conditions (Chen and Compans 1991), and then visualized
using a Phosphoimager (Molecular Dynamic)
Cell Transduction and Amplification by PCR TM
Jurkat cells, human CD4 + T-cells, were transfected with pCMV-Fabl 05-PE40 through electroporation, and were selected in medium containing G418 (800 μg / ml) for two to three weeks (Chen et al, 1994) Genomic DNA was extracted from the cells as described (Maniatis et al, 1986) The following oligonucleotides were used for the PCR ™ reactions pair A 5 -TTATTGCTAGCGTCGACCTTCGCGATGTA
CGGCCAG-3 '(SEQ ID NO 7) and 5 -GGTACCGAATTCTCTAGAACAA GATTTGGGCTC-3' (SEQ ID NO 8) par B 5 -GGTAGGCCTCAGG TGCAGCTGCAGGAG-3 '(SEQ ID NO 9) and 5 -TTTGCTAGCGG TATTATCATCGTG-3 (SEQ ID NO. 10) par C 5 -TTTGCGGCCGCGAA TTAATTCCGGTTA-3 (SEQ ID NO 11) and 5 '-TTTAAGATCTCCACAC TCTCCCCTGTTGAAGCT-3' (SEQ ID NO 12) par D 5'- TTGAATTCGGAGGTGGCGGAAGTCACCCTGGCG CGGAGTTC-3 '
(SEQ ID NO 13) and 5'-TTTATCGATTCTAGATTAC GGCGGTTT GCCGGGCTG-3 '(SEQ ID NO 14) PCR ™ products were analyzed on agarose gels
Enzyme-linked Immunosorbent Assay and ADP-Ribo assay
The ELISA assay was performed on plates coated with HIV-1 gp120 as described previously (Chen et al., 1994, incorporated herein by reference) Briefly, plates coated with HIV-1 gp120 were incubated with the culture medium from Jurkat-Fab105-PE40 or the Jurkat control, followed by the reaction with anti-human IgG ( Sigma) As shown in Figure 7A, the secreted Fab105-PE40 has specific binding activity to HIV-1 gp120. To further determine the binding activity, the ELISA assay was performed using other antibodies An ELISA microtiter plate was coated with 10 μg of recombinant HIV-1 gp120 (American Bio-Technologies Inc.) The plate was blocked with pH regulator TBST containing 1% BSA Jurkat-Fab105-PE40 or Jurkat control medium was added to the plate After goat anti-human immunoglobulin (Sigma) or anti-PEA (GIBCO BRL) in TBST was added to the cavities at a dilution of 1000, followed by an anti-goat IgG-peroxidase conjugate (Sigma) in TBST-1% of BSA at a dilution of 1 1000 detected one positive binding activity in the culture medium of Jurkat-Fab105-PE40 using either the anti-human IgG or anti-PEA, but no significant binding activity was not found in the control culture medium Jurkat
Cell Culture and HIV-1 Infection
To examine the cell killing activity of the cytotoxic cells, Costar-Transwell filters with a diameter of 12 mm, and a pore size of 40 μM, which could separate the cells, but not large molecules, were placed on plates of 12 cavities (Costar Corp, Cambridge, MA) and were used for the co-cultivation assay. The Jurkat cells of origin were infected with an HIV-1 virus from laboratory strain (IIIB) or two primary patient isolates ( INME and TPO), which were available to infect Jurkat cells and were obtained from Drs F and G Shaw at UAB (Birmingham, AL) Reverse transcpptase was measured every three to four days until RT reached its equilibrium Jurkat cells infected for HIV-1 were then placed in an upper chamber and the Jurkat-Fab105-PE40 cells and the Jurkat control cells were placed in the background chamber at different ratios. Viable cell numbers were counted daily. s of HIV-1 infected cells in the upper chambers The cell killing activity was expressed as percentage of viable infected cells co-cultured with Jurkat-Fab105-PE40 compared to viable infected cells co-cultured with the Jurkat control. effects of transduced cells in the production of HIV-1 RT, the primary HIV-1 isolate (WEAU) was used to infect Jurkat cells of origin On day six of post-infection a high level was detected
of RT activity in the culture medium of the infected Jurkat cell culture. These infected cells were then washed with PBS, resuspended in RPMI-1640/10% bovine serum medium at a cell density of 5x105 / ml. Infected Jurkat cells were then infected with Jurkat-Fab105-PE40 or the Jurkat control at different ratios RT tests were performed as previously described (Poiesz et al, 1980)
Construction of Specific Cytotoxic Cells Objective
Specific cytotoxic cells were generated in tumor by transducing an anti-HER2 / toxin expression vector into human lymphocytes. The genetically modified cells remained viable and expressed and secreted antibody / target toxins, since the interaction of the newly synthesized fusion toxins with EF-2 in the cytosol was blocked through the ER lipid membrane double layer and secretion vesicles The secreted antibody-toxin fusion proteins then recognized and destroyed target cells after internalization and release towards the cytosol of the Objective tumor cells However, the target toxin proteins did not annihilate the transduced toxin expression cells, which lack the target antigens on the surface of the cell. Thus this new class of cytotoxic cells with both targeted cytotoxicity aspects in the antibody as
cell-mediated will have broad applications for cancer and other disease therapies HIV-specific cytotoxic cells were generated by transducing an anti-HIV gp120 / toxin expression vector to human lymphocytes Genetically modified cells expressed and secreted the target antibody / toxin, that recognize and specifically kill target HIV-1 infected cells This antigen-specific cytotoxic cell functions as a producer of target toxin as well as a carrier, thus combining the advantages of antibody-mediated and cell-mediated immunotherapy
Specific Lymphocyte Protein Synthesis Reps Purpose
To determine if cellular protein synthesis is blocked in cells expressing the antibody / toxin protein, the 3H-leucine incorporation regimes of Jurkat-Fab105-PE40 cells, Jurkat control, MOLT-SFv23e-PE40 were examined or control MOLT The Jurkat-Fab105-PE40 or Jurkat control cells (0 5 x 106) were washed with RPMI 1640 medium and resuspended in 1 ml of RPMI 1640 medium containing 10% FBS. 3H-leuc was added Na (ICN Pharmaceuticals, Inc. 1 mCi / ml) to the medium at a final concentration of 4 μCi / ml, incubated for one hour at 37 ° C, followed by three washes with PBS for
Remove the unincorporated 3H-leucine After the precipitation and washing with tpcloroacetic acid (TCA), the pellets were resuspended in 4 ml of the synthetic liquid (ScmtiVerse BD, Fisher), and counted in a counter Synthalation (LS Beckman) Comparable levels of 3H-leucine incorporation were presented in Jurkat-Fab105-PE40 cells and Jurkat control cells
Timidine Incorporation Regimens of Specific Lymphocytes Objective
DNA synthesis regimes were determined by measuring thymidine incorporation Jurkat-Fab105-PE40 cells, Jurkat control, MOLT-Fab105-PE40 or MOLT control cells were washed with RPMI-1640, and resuspended in RPMI. -1640 / 10% FBS at a density of 0 5x106ml The cells were then added with or without PHA (Vector Laboratories) to a final concentration of 0 8 μg / ml for one hour. Then 10 μCi of (met? L-3H ) - t? m? d? na (ICN Pharmaceuticals, Inc., 1 mCi / ml) to each of the cell cultures, and incubated at 37 ° C for 12 hours. Cells were washed three times, and solubilized with 100 μl of 0 5% SDS The lysates of solubilized cells were then added to 4 ml of the synthesis fluid, and counted in a synthesizing counter. There was no significant difference in the incorporation of H-thymidine in Jurkat-Fab105-PE40 cells. or the Jurkat control In addition both cell lines were more than
95% viable as determined by exclusion of tppan blue Jurkat-Fab105-PE40 or Jurkat control cells were also found to have a similar proliferation curve Jurkat-Fab105-PE40 cells remained viable with normal proliferation during the observed period of four months The following examples are included to demonstrate the preferred embodiments of the invention. It should be appreciated by those skilled in the art that the techniques described in the following examples represent techniques discovered by the inventor to function well in the practice of the invention, can be considered which are preferred modes for their practice However, those skilled in the art should, in the light of the present description, appreciate that vain changes can be made in the specific modalities being described and that they continue to obtain a similar or similar result without departing of the spirit and scope of the invention
EXAMPLE 1 Anti-Tumor Cytotoxic Cells
The following example demonstrates the construction and use of a cytotoxic cell specific for HER-2 a transmembrane protein of the epidermal growth factor family, which is overexpressed in vain tumors of humans such as ovarian breast cancer. gastric and other types of
cancer (King et al, 1985, Slamon et al, 1989, Wen et al, 1992, Hynes, 1993, Muss et al, 1994) An individual anti- HER2 chain antibody (sFv) comprising the bound heavy chain variable region The light chain variable region derived from a monoclonal antibody 23e was found to have a high affinity binding activity to the extracellular domain of HER-2, and that can be efficiently internalized after binding (Batra et al, 1992, Kasprzyk et al, 1992, Bird et al, 1988, Marasco et al, 1993) In this study, the SFv23e gene with a leader signal sequence was used to fuse in frame with a truncated gene (PE40) encoding domains II (translocation) and III (catalytic) (amino acids 253 to 613) of PEA (Rosenberg et al, 1988, Rosenberg et al, 1990, Kawakami et al, 1994, Gary et al, 1984, Allured et al, 1986) under the control of a promoter. Cytomegalovirus (CMV) (Figure 1) This vector, pCMV-sFv23e-PE40, expresses and secretes the toxi Anti-HER2sFv-PE40 fusion proteins in mammalian cells An anterior primer for the amplification of the sFv23e gene with additional signal leader sequence including a HindIII cloning site was synthesized (5'-TTAAGCTTATGAAACATCTGTGGTTC TTCCTTCTCCTGGTGGCAGCTCCCAGATGGGTCCTGTCCGACGTCCA GCTGACC-3 (F-1) (SEQ ID NO 15) and an inverse primer with an additional Notl cloning site 5'-TTTGCGGCCGCGGAGAC GGTGACCGTGGT-3 (SEQ ID NO 16) were used to amplify the sFv23e gene with an additional leader signal sequence
The sFv23e gene with the leader sequence fragments was then cloned into HindllUNotl sites of the plasmid pRc / CMV (Invitrogen), and the resulting vector was designated pCMV-sFv23e-S The BglII-EcoR fragment containing the sequence encoding the amino acids (aa ) 253 to 613 of PEA was cut from plasmid pJH8 (ATCC), and cloned into vector pSP72 (Stratagene) (pSP-PE40) To incorporate a NotI site into the fragment, an initiator corresponding to amino acids 253 to 263 with a NotI site Additional (5'-CCCGCGGCCGCGCCGTCGCCGAGGAACTC-3 '(F-2) (SEQ ID NO: 17), and an inverse primer corresponding to amino acids 330 to 322 (5 -GCGGATCGCTTCGCCCAGGT-3' (SEQ ID NO: 18) were used to amplify DNA fragments encoding amino acids 253 to 322, and the amplified DNAs were then cut with NotUSall The Sal / Xba DNA fragment encoding amino acids 322 to 613 of PEA was cut and purified from the pSP-PE40 vector. NotUSall fragment of amino acid 253 / 322 and the Sall / Xbal fragment of amino acids 322/613 were then cloned into the NotUXbal sites of pCMV-sFv23e-S through a three-piece ligation. The resulting construct was identified through restriction enzyme digestion and was confirmed through DNA sequence analysis. To determine if mammalian cells are capable of producing the antibody / toxin fusion proteins, COS cells were transfected with pCMV-sFv23e-PE40 DNA and analyzed by immunofluorescent staining. staining
Strong positive in the cytoplasm, especially in the region of Golgí pepnuclear of the transfected cells was observed when the cells were incubated with either anti? -sFv23e antibody (Batra et al, 1992, Kasprzk et al, 1992, Bird et al, 1988 , Marasco et al, 1993), or anti-PEA (Gibco-BRL) indicating that transfected mammalian COS cells expressed a high level of anti-HERSsFv / PE40 fusion proteins This staining pattern resembles a pattern of typical secretion protein (Chen et al, 1994) Fluorescent-positive cells showed normal morphology, and over 98% of the transfected cells were viable, similar to control cells No significant staining was observed in the transfected control cells with the vector alone, and stained with a mixture of the anti? -sFv23e and -PEA antibodies. The expression of the fusion protein was further examined through radiolabeling and immunoprecipitation. cipitation COS cells were transfected with the pCMV-sFv23e-PE40 DNA or a control vector, and 60 hours later, the transfected cells were radiolabeled with 35S-c? ste? na for 4 hours (Chen and Compans, 1991). of antibody / toxin fusion approximately 70 kd, were immunoprecipitated from the culture medium of the translucent cells either through the anti? -sFv23e or anti i- PEA antibodies followed by the SDS-PAGE analysis No band was found of corresponding protein from the control cell These results confirmed that mammalian cells are capable
of producing antibody / toxin fusion proteins Cytotoxic cells specific for HER-2 were then generated by transduction of pCMV-sFv23e-PE40 to human T-cells (Molt-4 cells), followed by the selection of G418 (Chen et al. al, 1994) Human Imphocytes express undetectable levels of the HER-2 protein (Potter et al, 1989, Press et al, 1990) G418 resistant cells were collected and subcloned, and further analyzed for their biological aspects and expression of Protein Pulse-slot radiolabeling studies showed that fusion proteins were produced and secreted from transduced cells Genomic PCR ™ analyzes were used to detect whether the expression vector gene was incorporated Generic DNAs were isolated from cells of MOLT-sFv23e-PE40 and control of MOLT (Maniatis et al, 1986), and amplification was performed by PCR ™ The sFv23e DNA fragment (approximately 770 bp), domain II (approximately 450 bp) or the lll domain (approximately 610 bp) of the PEA DNA fragments were specifically amplified from the genomic DNA of MOLT-sFv23e-PE40 cells but not from the control cells In addition, a significant ADP-pbosylation activity was detected in the culture medium of MOLT-sFv23e-PE40, but only a background level of the ADP-pbosylation activity was found in the control cell medium indicating that the secreted fusion proteins have toxin enzymatic activity When compared to the
purified PEA activity (Gibco-BRL), more than 0 5 μg of the PEA protein was produced from MOLT-sFv23e-PE40 at 24 hours / 1 × 10 6 cells / ml. Vague biological aspects of MOLT-sFv23e-PE40 were examined First, when the cell proliferation and viability regimes of MOLT-sFv23e-PE40 and MOLT control cells were examined, there was no apparent difference Second, the DNA synthesis regimes of MOLT-sFv23e-PE40 cells and control MOLT were similar, as measured by the incorporation of 3H-t? m? d? na with or without PHA stimulation (0 8 μg / ml). Third, protein synthesis regimens of MOLT- cells sFv23e-PE40 and MOLT control were absolutely identical as measured by the incorporation of 3H-leucine In this way, the transduced MOLT-sFv23e-PE40 cells maintained their basic biological functions to determine if the transduced cells exhibit selective cytotoxicity to the cells from your mor objective, co-cultivation studies were carried out Cell lines expressing different levels of HER2 were co-cultured with MOLT-sFv23e-PE40 Imphocytes and MOLT control for 62 hours Significant cell annihilation was observed in the tumor cells (SKOV - 3 and N-87) over-expressing HER-2 (Batra et al, 1992, Kasprzyk et al 1992 Bird et al 1988, Marasco et al, 1993 Kraus et al 1987) co-cultured with MOLT-sFv23e-PE40, while that only a small percentage of cells were killed in the co-cultures
of MOLT-sFv23e-PE40 and the tumor cells (MCF-7 and NIH / 3T3) expressing low levels of HER-2 (Di Fiore et al, 1987) (FIG 3A) The MOLT control cells had no significant effects on the Tumor cells either expressing high or low levels of HER-2 Thus, protein synthesis of tumor cells by over-expressing HER-2 co-cultured with MOLT-sFv23e-PE40 was selectively inhibited 23e antibody also originating showed to inhibit cytotoxicity in a concentration dependent manner (Figure 3B) Collectively, these studies indicate that the transduced hnfocytes have selective cytotoxicity to the target cancer cells. The transformed nuclides, MOLT-sFv23e-PE40, and the control lymphocytes were generated through transfection of the plasmid DNA followed by G418 selection (Chen et al, 1994) The genomic DNAs were extracted from the cell lines as described (Maniatis, 1986) The oligonucleotides used for for PCR ™ reactions are listed below for A, F-1, and 5 -TTTAAGATCTACAGGAGACGGTGACCGTGG-3 '(SEQ ID NO 19) pair B F-2, and 5 -TTGCGGCCGCGAAAGGCGGCAGCCTGGCCGCG-3' (SEQ ID NO 5) for C 5 -GGTACCGAATTCTCTAGAGGCGACGTCA
GCTTCAGC-3 (SEQ ID NO 20), and 5 '-TTAATTGCGGCCGCTTACTT CAGGTCCTCGCG-3 (SEQ ID NO 21) DNA fragments amplified by PCR ™ were analyzed on agarose gels
EXAMPLE 2 In Vivo Activity of Anti-Tumor Cytotoxic Cells
The anti-tumor activity of the transduced cells described in Example 1 was further determined in a natural mouse model. The human gastric cancer cell line, N87, which has been shown to overexpress HER-2 proteins and grow well. As a subcutaneous tumor in a natural mouse (Batra et al, 1992, Kaspryzk et al, 1992, Bird et al, 1988, Marasco et al, 1993), the ability of adoptive transduced cells to infiltrate tissues with tumor was examined. natural athymic mice with xenograft grafts of N87 carcinoma were administered MOLT-sFv23e-PE40 cells through a vein injection in the tail 48 hours later, the mice were sacrificed and preparations of the tumor tissues were prepared and stained with an anti-PEA antibody followed by an anti-goat IgG conjugate (Sigma) Significant fluorescent staining was observed in the peripheral blood passage regions of the tumor tissues of the injected mice cured with MOLT-sFv23e-PE40, but not in animals injected with control cells Molt No apparent cytotoxicity was observed in normal tissues (liver, lung, heart, and heart) under the microscope The effects of tumor growth and survival of the animal were determined after MOLT-sFv23e-PE40 cells or Molt control cells (0 5 to 1 0x10) were intravenously injected to mice with
Neno tumor xenografts weekly for 6 weeks Administration of transduced cells strongly delayed growth of tumor xenografts (Figure 4A) Mice injected with Molt control cells all died in 70 days, but mice injected with MOLT -sFv23e-PE40 all survived during the observed period (Figure 4B). In this way, the administration of the transfected cells significantly inhibited tumor growth and prolonged the survival of the animal
EXAMPLE 3 Recombinant Anti-HIV-1 Toxin Fusion Protein Expression
To generate a specific HIV-1 cytotoxic cell, a human monoclonal neutralizing antibody (F105) was used against the CD4-binding site of HIV-1 gp120 expressed on the surface of HIV-1 infected cells (Sodroski et al, 1986 , Lifson et al, 1986) (Marasco et al, 1993, Thali et al, 1991, Chen et al, 1994) The gene (PE40) coding for domain II (for transubicación through the double membrane layer) and the domain lll (for adenosine diphosphate (ADP) -r? bos? lac? on EF-2) of PEA (Gray et al, 1984 Allured et al, 1986, Siegall et al, 1989) was fused to the chain gene K. of F105 (Chen et al 1994) Subsequently a bicistronic expression vector was constructed
pCMV-Fabl 05-PE40, which contains an Fd (VH + CH?) chain, an internal ribosome entry site sequence (IRES), and a chimeric? -PE40 gene (Figure 5) Cell-specific cytotoxic cells HIV-1 infected cells were generated through the transduction of the antibody / toxin expression vector to the human Jurkat CD4 + T lymphocytes, followed by the G418 selection. G418 resistant cells were collected and subcloned, and a genomic pohmerase chain (PCR ™) to detect if the Fab105-PE40 gene was incorporated into the cells As shown in Figure 6A, the CMV Fd promoter, Fd-IRES, IRES-K chain and the DNA fragments of domain III of toxin were specifically amplified from genomic DNA isolated from transduced Jurkat. Both Fd and -PE40 proteins were immunoprecipitated from Jurkat-Fab105-PE40 culture medium either through anti-human IgG or anti-PEA antibody. , i ndicating that the two fragments of Fd and the chain? -PE40 were joined together to the Fab fragments In this way, viable transduced lymphocytes are capable of producing and secreting the molecules of the anti-body-PE40 into the culture medium. Antigen binding of secreted Fab105-PE40 was examined through an enzyme-linked immunosorbent assay (ELISA). Positive binding activity was detected for gp120 in Jurkat-Fab105-PE40 culture medium, while none was detected. Significant binding activity in the control
Jurkat Compared to the binding activity of a dilution in sene of the purified F105 antibody (Chen et al, 1994), approximately 0 6 to 0 7 μg / ml of the Fab105-PE40 molecules were produced from Jurkat-Fab105 -PE40 (24 hr / 1x106 cells / ml) Enzymatic activity of Fab105-PE40 toxin was examined using an ADP-pbosylation assay Significant activity of ADP-pbosylation was detected in Jurkat-Fab105-PE40 culture medium, whereas only a background level of ADP-pbosylation activity was found in the control cell medium When compared to the ADP-pbosylation activity of purified PEA, approximately 0 8 μg / ml of the PEA protein was produced from of Jurkat-Fab105-PE40 (24 hr / 1x106 cells / ml) Several biological aspects of Jurkat-Fab105-PE40 were examined when the cell proliferation and the viability of Jurkat-Fab105-PE40 cells and Jurkat control cells examine In addition, the DNA synthesis regimes of Jurkat-Fab105-PE40 and Jurkat control cells were similar, as measured by the incorporation of JH-t? m? d? na with or without stimulation by PHA ( 0 8 μg / ml) The protein synthesis regimes of Jurkat-Fab105-PE40 cells and Jurkat control cells were also absolutely identical as measured through the incorporation of 3H-leucine. Fab105-PE40 transduced maintained their basic biological functions
Selective Cytotoxicity to Cells Infected by HIV-1
The selective cytotoxicity in HIV-1 infection of transduced Jurkat-Fab105-PE40 cells was evaluated using co-cultivation assays to examine cell killing activity against HIV-1 infected cells and effects on reverse transcpptase production viral (RT) To examine the cell killing activity of the cytotoxic cells, Jurkat T lymphocytes of origin were infected with an HIV-1 laboratory strain (HIB) or two primary HIV-1 isolates (INME and TPO), respectively, and then co-cultured with Jurkat-Fab105-PE40 or Jurkat control cells at different ratios on Costar-Transwell filter plates with twelve wells. The viable HIV-1 infected cells in the upper chamber were counted, and the percentage of non-viable cells is shown in Figure 7A Significant annihilation of cells infected with strain IIIB or primary HIV-1 isolates was observed in co-cultures c on Jurkat-Fab105-PE40 but not with the Jurkat control No adverse effects were observed in non-infected Jurkat cells To further observe infection with viruses in the co-cultures Jurkat cells of origin were infected with a primary isolate of patient with HIV (WEAU) for 6 days, washed with PBS three times and then co-cultured with Jurkat-Fab105-PE40 cells or Jurkat control cells at different ratios.
HIV-1 infection in the co-culture was verified every 3 to 4 days by measuring the viral RT activity. As shown in Figure 7B, in the co-culture with Jurkat-Fab105-PE40, only low levels of RT were observed. Throughout the period, while significantly higher levels of RT were detected in the co-culture with the Jurkat control cells When increasing ratios of Jurkat-Fab105-PE40 cells were used to target HIV-1 infected cells, a stronger inhibition of HIV-1 infection Jurkat-Fab105-PE40 cells were not found to have any adverse effect on Jurkat co-cultured and other lymphocyte lines not infected with HIV-1 Thus, Jurkat-Fab105-PE40 effectively inhibited HIV infection, probably by selective cytotoxicity and neutralization activities of Fab105-PE40 fusion proteins
EXAMPLE 4 Generation of Specific Annihilation Cells of B Cell Lineage
Recently, a single mupno monoclonal antibody Lym-1 was generated, which recognizes a polymorphic vanant of the HLA-Dr antigen present on the surface of the cell of abnormal and malignant B cells (Epstein et al, 1987, Hu et al 1995). antibody, Lym-1 is remarkably a specific B cell, with significantly increased activity for the cells of
lymphoma, when compared to normal B lymphocytes Lymphoma-1 123l-labeled imaging studies have shown selective localization at lymphoma sites, as well as an ability to detect tumor sites (Epstein et al, 1985, 1987, DeNardo et al, 1987, 1988a, 1988b, Hu et al, 1989) No evidence of Lym-1 antibody bound to T cells or other normal cells and tissues was shown as demonstrated by the immunochemical detection of tissue sections and radio formation Image (Epstein et al, 1987) In summary, Lym-1 antibody has been shown to be specific to malignant B cells, and can be used for objective therapy for B lineage disorder in order to take advantage of the specific character of the antibody Lym-1, specific annihilation cells were generated for B-lineage / lymphoma leukemia through transduction with chimeric gene Transduced cells recognized and annihilated leukemia / lymphoma cells of lineage B Specifically, the antibody variable region genes of the Lym-1 antibody were cloned and assembled to an individual chain antibody gene (sFv). The sFv-Lym gene was fused in frame to the domain sequence ll-lll of PEA, and cloned into a mammalian expression vector Specific cytotoxic T lymphocytes, which are capable of producing and secreting the sFv-Lym / toxin fusion protein, were generated through the transduction of the sFv-Lym / toxin gene with a signal peptide leader at 11 nf oc i tos human T The cell binding
B and the toxin catalytic activities of the secreted antibody / toxin molecules were demonstrated through the ADP-pbocylation assay and the flow cyrometpco analysis. The selective cytotoxicity of the transduced cytotoxic cells to the leukemia / B-cell lymphoma cells were observed m vitro The results demonstrate that this new class of cytotoxic cells with specific defined character can have wide applications for the treatment of disorders of lineage B and other types of cancer
Construction and Expression of Antibody / Toxin Fusion Proteins
The cDNA genes of the Lym-1 monoclonal antibody were obtained from Dr A Epstein at Univ Caf San Diego (Epstein et al, 1987). The VH and V cDNA genes were amplified by
PCR ™, respectively, and then gel-purified A PCR ™ extension was then used to assemble VH and VL to sFv The resulting sFv-Lym protein consists of light and heavy chain variable regions of the Lym-1 antibody bound through a peptide with a leader signal peptide sequence The PEA gene obtained from ATCC contains three functional domains
Domain I, cell recognition Domain II, trans-localization domain (amino acid residues (aa) 253-404), and domain lll catalytic domain (aa 405-613) (Pastan 1992)
Specifically, the variable region genes of the Lym-1 antibody were used as templates for PCR ™ reactions as previously described (Chem et al, 1994) An anterior primer for VH-Lym with an additional signal peptide leader sequence and a site of cloning HmdlW 5'-TTAAGCTTCATATGGAACATC TGTGGTTCTTCCTTCTCCTG GTGGC AGCTCCCAG ATGG GTC CTGTCC-3 'SEQ ID NO 22 (F-1)), and a reverse primer with an additional interchain link sequence 5'-GCTCCCACCACCTCCGGAG CCACCGCCACCTGCAGAGACGTGACCCAGAGT-3', SEQ ID NO 23, were used to amplify the VH gene with a leader signal peptide sequence. An anterior primer for VL-Lym with an additional interchain sequencer sequence 5 - GGTGGCGGTGGCTCCGGAGGTGGTGGTGGGAGCGGTGGCGGCGG ATCTGAGCTTCGTGAATGACCCAGTCTCCA-3 'SEQ ID NO 24, and an inverse primer with a Notl cloning site, 5'-AAAGCGGCCGCACGTTTGATCTCCAGCTTGGT-3 'SEQ ID NO 25, (F-2) were used to amplify the VH gene by a sequence of leader signal peptide VH and VL DNA fragments amplified by PCR ™ were then assembled to sFv-Lym through extension PCR ™ using primers F-1 and F-2 sFv-Lym with leader sequence fragments were then cloned to the Hind l Notl sites of plasmid pRc / CMV (Invitrogen) and the resulting vector were designated pCMV-sFv-Lym
Generation of Specific Cytotoxic Cells for Leukemia / Lineage B Lymphomas
Cytotoxic cells specific for lineage B cells were generated through transduction of the fused antibody / toxin expression vector The methods for gene transfer and selection were previously described (Chen et al, 1994, 1995) In summary, the lymphocytes T humans such as Molt, or SupT, were transfected with the antibody / toxin expression DNA through electroporation, and were selected in a culture medium containing G418. To determine the expression of recombinant protein, the transduced cells were radiolabelled and immunoprecipitated. as previously described Samples were then analyzed through SDS-PAGE, and visualized through a Phospholmager PCR ™ analysis of the genomic DNA of the transduced hnfocytes was used to determine if the antibody / toxin gene was incorporated into the gene of the transduced nuclides The genomic DNAs were extracted from the transduced lymphocytes s according to a standard method (Maniatis et al, 1986) The oligonucleotides used for the PCR ™ reactions, which correspond to sFv-Lym and the PEA toxin gene, are listed below for A primer F-1 and 5 '- TTTAAGATCTACAGGAGA CGGTGACCGTGG-3 (SEQ ID NO 26), pair B F-2 and 5 -TTGCGGCCGCGAAAGGCGGCAGCCTGGCCGCG-3 (SEQ ID NO 27)
pair C 5 -GGTACCGAATTCTCTAGAGGCGACGTCAGCTTCAGC-3 '(SEQ ID NO 28), and 5 -TTAATTGCGGCCGCTTACTTCAGGTCCTCGCG-3' (SEQ ID NO 29) PCR ™ products were analyzed through agarose gel electrophoresis Imfocyte genomes that incorporated DNA which encodes SfV-Lym / toxma is expected to generate three size-specific bands with the PCR ™ primer pairs as follows: sFv-Lym DNA fragment of approximately 770 bp, a PEA domain II of approximately 450 bp and an Ill domain of PEA of approximately 610 bp To examine the expression of antibody / toxin in mammalian cells, a passenger expression assay was performed COS-1 cells were developed on coverslips, and 5 μg of the antibody / toxin expression plasmid cDNAs were transfected. to the cells using pofectma (Chen and Compans, 1992) After a 48-hour incubation, the cells were fixed and stained with an anti-toxin antibody (Gibco-BRL), followed by a conjugate flu. Orescent Strong positive fluorescent staining was observed in the transfected cells while no significant staining was found in the control cells Fluorescent staining is located across the cytoplasm and Golgí pepnuclear region representing a typical secretion protein staining pattern. it is evident that the transfected cells expressing the antibody / toxin proteins maintain their normal morphology, suggesting that mammalian cells are capable of producing the
antibody / toxin, while remaining viable
B-Cell Linkage and Secretory Recombinant Antibody / Toxin Fusion Protein Toxin Catalyst Activities
To determine whether the antibody fusion proteins / secreted toxins maintain the antigen-binding activity, cytomatic flux analyzes were performed on malignant B-cell lines such as, Ravi Ravi cells (1 x 106) (ATCC) were coated with different Seen dilutions of the antibody / toxin fusion proteins at 4 ° C for 30 minutes, and then incubated with an anti-mouse IgG antibody conjugate or an anti-PEA antibody followed by a labeled antibody conjugate (Sigma) for a period and under conditions that favor the development of additional immunocomplex formation (eg, incubation for 2 hours at room temperature in a solution containing PBS such as PBS-Tween) T-cell lines such as Molt-4, SupT , were used as a negative control A positive result indicates that the antibody / toxin fusion protein produced by the lymphocytes is able to bind to B cells
EXAMPLE 5 Production of Target Toxins Via Brain Cells
TGF-PE40 fusion toxins were transfected expressing plasmid DNA to normal neurons through hposome encapsulation and the TGF-PE40 fusion toxins were produced and secreted from the neurons In a natural mouse model of tumor xenograft , transfected neurons were found to express TGF-PE40 fusion proteins after one month of transfection Plasmid TGF-PE40 DNAs were also transfected into brain tumor cells in a tissue culture and in natural mouse models of xenograft Tumor We found that transfected tumor cells express TGF-PE40, which results in the death of surrounding tumor cells with an amplified cytotoxicity, i.e., a transfected cell is able to kill many surrounding tumor cells In this way, target toxins can be used locally to produce and amplify a selective cell killing ability, which s may have broad implications for the treatment of cancer and autoimmune diseases, and particularly for the treatment of cancer in the brain. For example, after a tumor is removed from the brain, the plasmid / liposomes may be administered to the subject's brain. Immunotoxins produced at that time may be able to direct and destroy the cells of
tumor left by surgery that could not otherwise be detected until they are grown in another tumor The present invention therefore provides a powerful therapy for the treatment of tumors in the brain
EXAMPLE 6 Generation of Auxiliary Cells T of Annihilating Cells
Specific Associated with Autoimmune Diseases or Reactions
To transduce T lymphocytes so as to produce immunotoxins that are capable of recognizing antigens specifically associated with a subset of T helper cells associated with an autoimmune disease or reaction such as arthritis, a blood sample, of approximately 200 cc / sample, was isolated from the subject The lymphocytes were isolated from the blood sample and developed under appropriate conditions following standard protocols as illustrated by Janda et al, Manual of Clinical Microbiology, 5th Edition, American Society for Microbiology, Washington, DC, Chapter 19, p 137, ( incorporated herein by reference) In this manner, approximately 1011 lymphocytes can be isolated from the culture after approximately two weeks. The isolated cultured hnfocytes are transduced as described above so that they can produce and secrete.
an immunoreactive cytotoxin with the desired species of T helper cells These lymphocytes can then be re-infused, injected or returned to the host subject in a pharmaceutically acceptable carrier so that a dose of approximately 109 hnfocytes is delivered. The dose can be administered at intervals that They vary from two weeks to six months or as desired. The site of choice for administration can be waived as appropriate to the condition being treated. For example, in the case of inflammatory arthritis of a joint, it may be desirable to administer the dose to the joint Subcutaneous, intramuscular, intradermal, or other sites may be used as appropriate All the compositions and methods described and claimed herein may be made and executed without undue experimentation in the light of the present disclosure, since the compositions and methods of this invention have been described in In accordance with the terms of preferred embodiments, it will be apparent to those skilled in the art that variations may be applied to the compositions and / or methods and to the steps or sequence of method steps described herein without departing from the concept, spirit and scope of the invention. The invention More specifically it will be apparent that certain agents that are both chemically and physiologically related can be substituted by the agents described herein while similar or equal results can be obtained. All these substitutions and modifications
similar obvious to those skilled in the art are intended to be within the spirit, scope and concept of the invention as defined by the appended claims
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The following references, to the extent that they provide illustrative procedures or other supplementary details to those set forth herein, are specifically incorporated herein by reference Allured, Collier, Carroll, and McKay, "Structure of exotoxm A of
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LIST OF SEQUENCES
(1) GENERAL INFORMATION (i) APPLICANT (A) NAME Wake Forest University (B) CALLE Medical Center Boulevard (C) Wmston-Salem CITY (D) STATE North Carolina (E) COUNTRY USA (F) ZIP CODE (AREA) 27157 -1023 (G) TELEPHONE (512) 418-3000 (H) TELEFAX (512) 474-7577 (II) TITLE OF THE INVENTION CITOTOXIC CELLS OBJECTIVE (ni) NUMBER OF SEQUENCES 29 (iv) LEGIBLE FORM BY COMPUTER (A) TYPE MEDIUM flexible disk (B) COMPUTER IBM PC compatible (C) PC-DOS / MS-DOS OPERATING SYSTEM (D) SOFTWARES Patentln Relay # 1 0, Version # 1 30
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(2) INFORMATION FOR SEC ID NO 1 (i) SEQUENCE CHARACTERISTICS (A) LENGTH 24 base pairs (B) TYPE nucleic acid (C) SINGLE CHAIN STRUCTURE (D) Linear TOPOLOGY (xi) SEQUENCE DESCRIPTION SEC ID NO 1 TTTGCTAGCG GTATTATCAT CGTG 24
(2) INFORMATION FOR SEC ID NO 2 (i) SEQUENCE CHARACTERISTICS (A) LENGTH 2 base pairs (B) TYPE nucleic acid (C) SINGLE CHAIN STRUCTURE (D) Linear TOPOLOGY (xi) SEQUENCE DESCRIPTION SEC ID NO 2 TTTGCGGCCG CGAATTAATT CCGGTTA 27
(2) INFORMATION FOR SEQ ID NO 3 (i) SEQUENCE CHARACTERISTICS (A) LENGTH 28 base pairs (B) TYPE nucleic acid (C) SINGLE CHAIN STRUCTURE (D) Linear TOPOLOGY (xi) SEQUENCE DESCRIPTION SEC ID NO 3
GGTTGCTGC ATGGAAACCC CAGCGCAG 28
(2) INFORMATION FOR SEQ ID NO 4 (i) SEQUENCE CHARACTERISTICS (A) LENGTH 31 base pairs (B) TYPE nucleic acid (C) SINGLE CHAIN STRUCTURE (D) Linear TOPOLOGY (xi) SEQUENCE DESCRIPTION SEC ID NO 4 AAAATCTAGA TTAACACTCT CCCCTGTTGA AT 31
(2) INFORMATION FOR SEC ID NO 5 (i) SEQUENCE CHARACTERISTICS (A) LENGTH 32 base pairs (B) TYPE nucleic acid (C) SINGLE CHAIN STRUCTURE (D) Linear TOPOLOGY (xi) SEQUENCE DESCRIPTION SEC ID NO 5 TTGCGGCCGC GAAAGGCGGC AGCCTGGCCG CG 32
(2) INFORMATION FOR SEQ ID NO 6 (i) SEQUENCE CHARACTERISTICS (A) LENGTH 20 base pairs (B) TYPE nucleic acid (C) SINGLE CHAIN STRUCTURE
(D) Linear TOPOLOGY (xi) SEQUENCE DESCRIPTION SEQ ID NO 6 GCGGATCGCT TCGCCCAGGT 20
(2) INFORMATION FOR SEQ ID NO 7 (i) SEQUENCE CHARACTERISTICS (A) LENGTH 37 base pairs (B) TYPE nucleic acid (C) SINGLE CHAIN STRUCTURE (D) Linear TOPOLOGY (xi) SEQUENCE DESCRIPTION SEC ID NO 7 TTATTGCTAG CGTCGACCTT CGCGATGTAC GGGCCAG 37
(2) INFORMATION FOR SEQ ID NO 8 (i) SEQUENCE CHARACTERISTICS (A) LENGTH 33 base pairs (B) TYPE nucleic acid (C) SINGLE CHAIN STRUCTURE (D) Linear TOPOLOGY (xi) SEQUENCE DESCRIPTION SEC ID NO 8 GGTACCGAAT TCTCTAGAAC AAGATTTGGG CTC 33
(2) INFORMATION FOR SEC ID NO 9 (i) SEQUENCE CHARACTERISTICS (A) LENGTH 27 base pairs
(B) TYPE nucleic acid (C) SINGLE CHAIN STRUCTURE (D) Linear TOPOLOGY (xi) SEQUENCE DESCRIPTION SEQ ID NO 9 GGTAGGCCTC AGGTGCAGCT GCAGGAG 27
(2) INFORMATION FOR SEQ ID NO 10 (i) SEQUENCE CHARACTERISTICS (A) LENGTH 24 base pairs (B) TYPE nucleic acid (C) SINGLE CHAIN STRUCTURE (D) Linear TOPOLOGY (xi) SEQUENCE DESCRIPTION SEC ID NO 10 TTTGCTAGCG GTATTATCAT CGTG 24
(2) INFORMATION FOR SEQ ID NO 11 (i) SEQUENCE CHARACTERISTICS (A) LENGTH 27 base pairs (B) TYPE nucleic acid (C) SINGLE CHAIN STRUCTURE (D) Linear TOPOLOGY (xi) SEQUENCE DESCRIPTION SEC ID NO 11 TTTGCGGCCG CGAATTAATT CCGGTTA 27
(2) INFORMATION FOR SEC ID NO 12
(i) SEQUENCE CHARACTERISTICS (A) LENGTH 33 base pairs (B) TYPE nucleic acid (C) SINGLE CHAIN STRUCTURE (D) Linear TOPOLOGY (xi) SEQUENCE DESCRIPTION SEC ID NO 12 TTTAAGATCT CCACACTCTC CCCTGTTGAA GCT 33
(2) INFORMATION FOR SEQ ID NO 13 (i) SEQUENCE CHARACTERISTICS (A) LENGTH 41 base pairs (B) TYPE nucleic acid (C) SINGLE CHAIN STRUCTURE (D) Linear TOPOLOGY (xi) SEQUENCE DESCRIPTION SEC ID NO 13 TTGAATTCGG AGGTGGCGGA AGTCACCCTG GCGCGGAGTT C 41
(2) INFORMATION FOR SEQ ID NO 14 (i) SEQUENCE CHARACTERISTICS (A) LENGTH 36 base pairs (B) TYPE nucleic acid (C) SINGLE CHAIN STRUCTURE (D) Linear TOPOLOGY (x?) SEQUENCE DESCRIPTION SEC ID NO 14 TTTATCGATT CTAGATTACG GCGGTTTGCC GGGCTG 36
(2) INFORMATION FOR SEC ID NO 15 (i) SEQUENCE CHARACTERISTICS (A) LENGTH 80 base pairs (B) TYPE nucleic acid (C) SINGLE CHAIN STRUCTURE (D) Linear TOPOLOGY (xi) SEQUENCE DESCRIPTION SEC ID NO 15 TTAAGCTTAT GAAACATCTG TGGTTCTTCC TTCTCCTGGT GGCAGCTCC GGCAGCTCCC AGATGGGTC 60 TGTOCGaG3TCaaarn3aCC 80
(2) INFORMATION FOR SEQ ID NO 16 (i) SEQUENCE CHARACTERISTICS (A) LENGTH 29 base pairs (B) TYPE nucleic acid (C) SINGLE CHAIN STRUCTURE (D) Linear TOPOLOGY (xi) SEQUENCE DESCRIPTION SEC ID NO 16 TTTGCGGCCG CGGAGACGGT GACCGTGGT 29
(2) INFORMATION FOR SEC ID NO 17 (i) SEQUENCE CHARACTERISTICS (A) LENGTH 29 base pairs (B) TYPE nucleic acid (C) SINGLE CHAIN STRUCTURE (D) Linear TOPOLOGY
(xi) SEQUENCE DESCRIPTION SEC ID NO 17 CCCGCGGCCG CGCCGTCGCC GAGGAACTC 29
(2) INFORMATION FOR SEQ ID NO 18 (i) SEQUENCE CHARACTERISTICS (A) LENGTH 20 base pairs (B) TYPE nucleic acid (C) SINGLE CHAIN STRUCTURE (D) Linear TOPOLOGY (xi) SEQUENCE DESCRIPTION SEC ID NO 18 GCGGATCGCT TCGCCCAGGT 20
(2) INFORMATION FOR SEQ ID NO. 19 (i) SEQUENCE CHARACTERISTICS (A) LENGTH 30 base pairs (B) TYPE nucleic acid (C) SINGLE CHAIN STRUCTURE (D) Linear TOPOLOGY (xi) SEQUENCE DESCRIPTION SEC ID NO 19 TTTAAGATCT ACAGGAGACG GTGACCGTGG 30
(2) INFORMATION FOR SEC ID NO 20 (i) SEQUENCE CHARACTERISTICS (A) LENGTH 26 base pairs (B) TYPE nucleic acid
(C) Individual CHAIN STRUCTURE (D) Linear TOPOLOGY (xi) SEQUENCE DESCRIPTION SEC ID NO 20 GGTACCGAAT TCTCTAGAGG CGACGTCAGC TTCAGC 36
(2) INFORMATION FOR SEQ ID NO 21 (i) SEQUENCE CHARACTERISTICS (A) LENGTH 32 base pairs (B) TYPE nucleic acid (C) SINGLE CHAIN STRUCTURE (D) Linear TOPOLOGY (xi) SEQUENCE DESCRIPTION SEC ID NO 21 TTAATTGCGG CCGCTTACTT CAGGTCCTCG CG 32
(2) INFORMATION FOR SEQ ID NO 22 (i) SEQUENCE CHARACTERISTICS (A) LENGTH 68 base pairs (B) TYPE nucleic acid (C) SINGLE CHAIN STRUCTURE (D) Linear TOPOLOGY (xi) SEQUENCE DESCRIPTION SEC ID NO 22 TTAAGCTTCA TATGGAACAT CTGTGGTTCT TCCTTCTCCT GGTGGCAGCT CCCAGATGG 60 TCCTGTCC 68
(2) INFORMATION FOR SEC ID NO 23
(i) SEQUENCE CHARACTERISTICS (A) LENGTH 51 base pairs (B) TYPE nucleic acid (C) SINGLE CHAIN STRUCTURE (D) Linear TOPOLOGY (xi) SEQUENCE DESCRIPTION SEC ID NO 23 GCTCCCACCA CCTCCGGAGC CACCGCCACC TGCAGAGACG TGACCAGAG T 51
(2) INFORMATION FOR SEC ID NO 24 (i) SEQUENCE CHARACTERISTICS (A) LENGTH 71 base pairs (B) TYPE nucleic acid (C) SINGLE CHAIN STRUCTURE (D) Linear TOPOLOGY (xi) SEQUENCE DESCRIPTION SEC ID NO 24 GGTGGCGGTGGCTCCO3G? GGTGGTGGGAGCGGTGGO3G8 60
CCCAGTCTCC A 71
(2) INFORMATION FOR SEC ID NO 25 (i) SEQUENCE CHARACTERISTICS (A) LENGTH 32 base pairs (B) TYPE nucleic acid (C) SINGLE CHAIN STRUCTURE (D) Linear TOPOLOGY (xi) SEQUENCE DESCRIPTION SEC ID NO 25
AAAGCGGCCG CACGTTTGAT CTCCAGCTTG GT 32
(2) INFORMATION FOR SEC ID NO 26 (i) SEQUENCE CHARACTERISTICS (A) LENGTH 30 base pairs (B) TYPE nucleic acid (C) SINGLE CHAIN STRUCTURE (D) Linear TOPOLOGY (xi) SEQUENCE DESCRIPTION SEC ID NO 26 TTTAAGATCT ACAGGAGACG GTGACCGTGG 30
(2) INFORMATION FOR SEC ID NO 27 (i) SEQUENCE CHARACTERISTICS (A) LENGTH 32 base pairs (B) TYPE nucleic acid (C) SINGLE CHAIN STRUCTURE (D) Linear TOPOLOGY (xi) SEQUENCE DESCRIPTION SEC ID NO 27 TTGCGGCCGC GAAAGGCGGC AGCCTGGCCG CG 32
(2) INFORMATION FOR SEQ ID NO 28 (i) SEQUENCE CHARACTERISTICS (A) LENGTH 36 base pairs (B) TYPE of nucleic acid (C) SINGLE CHAIN STRUCTURE
(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28: GGTACCGAAT TCTCTAGAGG CGACGTCAGC TTCAGC 36
(2) INFORMATION FOR SEQ ID NO: 29: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 32 base pairs (B) TYPE: nucleic acid (C) CHAIN STRUCTURE: individual (D) TOPOLOGY: linear (xi) ) SEQUENCE DESCRIPTION: SEQ ID NO: 29: TTAATTGCGG CCGCTTACTT CAGGTCCTCG CG 32
Claims (1)
- CLAIMS 1 A mammalian cell that expresses and secretes an immunotoxin 2 A mammalian cell according to claim 1, further defined as a cytotoxic lymphocyte 3 A mammalian cell according to claim 1, further defined as a neuron mammal according to claim 1, wherein said immunotoxin comprises an anti-tumor antibody domain of a mammalian cell according to claim 3, wherein said anti-tumor antibody domain is immunoreactive with a HER2 antigen. of mammal according to claim 3, wherein said antibody domain is immunoreactive with a Lym-1 antigen. A mammalian cell according to claim 1, wherein the antibody domain of said immunotoxin is immunoreactive with a viral antigen. 8 A mammalian cell according to claim 7 wherein the viral antigen is an HIV antigen 9 A lymph Cytotoxic cyto T according to claim 2, wherein the antibody domain of the immunotoxin is immunoreactive with an auxiliary cell T A mammalian cell according to claim 1, wherein the toxin of the immunotoxin is one or more domains of a Pseudomonas exotoxin, diphtheria toxin, nema A, abpna, gelonin or sapopne toxin. 11 A mammalian cell according to with claim 1, wherein the immunotoxin comprises a Pseudomonas exotoxin domain. A mammalian cell according to claim 1, dispersed in a pharmaceutically acceptable carrier solution. A mammalian cell transfected with an expression vector, wherein vector expresses a fusion protein comprising a leader sequence and an immunotoxin, wherein said leader directs the immunotoxin to the endoplasmic reticulum of the cell. A cell according to claim 13, wherein the vector is a plasmid. according to claim 13, wherein the vector is a viral vector 16 A cell according to claim 1 3, wherein the immunotoxin immunoreacts with an antigen associated with the tumor 17 A cell according to claim 13, wherein the immunotoxin immunoreacts with a viral associated antigen 18 A cell according to claim 13, wherein the immunotoxin immunoreacts with an antigen associated with the T 19 helper cell. A cell according to claim 17, wherein the viral associated antigen is an HIV-associated antigen. A cell according to claim 19, wherein wherein the associated antigen to HIV is a gp120 antigen. A cell according to claim 16, wherein the tumor associated antigen is a HER2 antigen. A cell according to claim 16, wherein the tumor associated antigen is a Lym-1 antigen. cell according to claim 13, further defined as a cytotoxic hnfocyte. A cell according to claim 13, further defined as a neuron. A pharmaceutical composition comprising a cell according to claim 13, dispersed in a pharmaceutically acceptable carrier. 26 A method for killing a cancer cell comprising contacting said cancer cell A method according to claim 26 wherein the mammalian cell is a cytotoxic lymphocyte. according to claim 26 wherein the cancer cell on expresses HER-2. A method according to claim 26, wherein the mammalian cell is a neuron. A method according to claim 26, wherein the cancer cell on expresses Lym-1. according to claim 28, wherein the cancer cell is a breast cancer cell, ovary or gastric A method according to claim 26, wherein the cancer cell is a brain cancer cell 33 A method according to claim 26, wherein wherein the cancer cell is in an animal subject and said mammalian cell is administered to said subject in a pharmaceutical composition. A method according to claim 33, wherein the subject is a human patient with cancer. A method for killing a virally infected cell comprising contacting said cell with an immunotoxin expressed from a cytotoxic T cell, wherein the antibody portion of the immunotoxin recognizes the antigen expressed by said virally infected cell. A method according to claim 35, wherein the cytotoxic T cell is administered in a solution. pharmaceutical to an animal subject that has a virally infected cell 37 A method according to the law vindication 36, where the subject is a human 38 A method for inhibiting an HIV infection in a subject comprising administering a pharmaceutical solution to said subject, wherein the solution comprises a cytotoxic T cell that expresses and secretes an anti-HIV immunotoxin. inhibit tumor cell growth in a subject comprising administering a cytotoxic T cell by expressing an anti-tumor cell immunotoxin to said subject. A method for producing a recombinant immunotoxin comprising the steps of obtaining a mammalian cell transfected with a mammalian vector. expression, wherein the expression vector expresses a fusion protein comprising a leader sequence and a mmunotoxin, and wherein the leader directs the immunotoxin to the endoplasmic reticulum of the cell, and culturing the cell under conditions effective to express the immunotoxin. The method according to claim 40, further comprising the step of Islar said immunotoxin
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US740003 | 1991-08-02 |
Publications (1)
Publication Number | Publication Date |
---|---|
MXPA99003758A true MXPA99003758A (en) | 2000-04-24 |
Family
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