JP2001523731A - Proliferatively active products for treating growing cells - Google Patents

Abstract

  (57) [Summary] Production comprising a proliferative active ingredient, typically a cytokine or growth factor, linked to a biologically active agent that has a high affinity receptor and acts preferentially or selectively on proliferating cells object. For example, an active promoter for growth, such as active IL-2, can be beneficially used to supply a pharmacologically desired species to cells where growth is not desired. For example, some medicaments of the invention utilize a molecule containing a growth activity promoter to control or inhibit growth. Biologically active agents are usually selected from the group comprising antiproliferative agents, compounds that interfere with nucleotide synthesis, radioisotopes, gene sequences and antisense nucleotide sequences.

Description

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the use of proliferatively active compounds, in particular cytokines or growth factors, as active vectors for pharmacologically active compounds such as, for example, generic drugs or genes.

[0002] The proliferation, differentiation and functional activities of many cells, especially hematopoietic and immune cells, are controlled by proteins called cytokines and growth factors. Because of the overlapping mechanisms and effects on the two groups of target cells, it is difficult to distinguish between them. For example, the cytokine interleukin-2 is also known as a T cell growth factor. Both cytokines and growth factors are peptidic hormones.

[0003] More specifically, cytokines control the functional state of their target cells (eg, they can promote or suppress both quantitatively and qualitatively), while growth factors control their target cell lineage. Is more focused on promoting, controlling and maintaining growth, differentiation and survival.

[0004] Both cytokines and growth factors recognize specific membrane receptors that are unique to the particular cytokine or growth factor present on its target cells. In other words, each receptor can express dynamic binding activity to its specific cytokine or growth factor based on physiological and / or pathological conditions. These receptors are low, medium,
Alternatively, it can be classified as high affinity. Most importantly, only high-affinity receptors can recognize, capture, and internalize their associated cytokines or growth factors. For these receptors and their ligands,
This will be described in more detail below with reference to cytokines.

[0005] Cytokines are a group of molecules other than antibodies produced from lymph cells that are involved in signaling between cells of the immune system to promote or suppress cell functions. Cytokines often transmit their effects through specific receptors expressed on target cells. Cytokines are glycosylated or non-glycosylated polypeptides, and T cells are considered the primary source of cell-mediated reactions, but both T cells and B cells can secrete. The complexity of cytokine studies is due to the fact that cytokines no longer function in vivo in the isolated state. This is exemplified by the observation that the effects of many cytokines are synergistic. Important cytokines include interleukin (IL), tumor necrosis factor (TNF) and interferon (IFN). Furthermore, various colony stimulating factors (CSF) are secreted from myeloid erythroid cells.

At present, receptors for various cytokines have been cloned and their structures (amino acid sequences) have been analyzed. As a result, many of them could be put together into superfamilies according to their common homology region on the primary structure. For the purposes of the present invention, the main superfamily is the cytokine receptor superfamily (sometimes called the hematopoietic receptor superfamily).
per family; CKR-SF) and the interferon receptor superfamily (interferon
receptor super family; IFNR-SF) (Reference material 1). The term "superfamily" should be used only when describing proteins having 50% or less amino acid sequence homology. 50% amino acid sequence homology
The proteins beyond are termed "families."

[0007] Many cytokine and growth factor receptors have various domains or combinations of repeats. A domain is a protein sequence or fragment that forms discrete structural units that can capture and / or convert specific signals. For the purposes of the present invention, the desired domain is an extracellular region (a region located on the surface of the target cell lineage in question). Studies targeting receptor binding have shown that multiple CKR-SF members (or hematopoietic receptor superfamily) have one or more binding affinities. Typically, these sites are low (ie, 1-10) for a particular ligand (cytokine or growth factor).
mM) or high (i.e., at least 1 pM, usually 10-100 pM). For most of these receptor complexes, additional subunits required for high affinity receptor expression have been identified. These subunits (also called affinity converters or converter chains) may be expressed on the cell surface after a given activating or inhibitory stimulus has been applied through the receptor ligand. Many. As a result, action is enhanced only in cells with high affinity receptors, not in resting cells (usually with low activity receptor complexes), and involvement of local / regional / systemic networks. Provides a physiological basis for unaccompanied paracrine stimulation / inhibition.

[0008] That is, in order to transmit an immune response, T cells must change from a resting state to an active state. T cells stimulated by foreign antigens enter a program of cell activation that leads to de novo synthesis of IL-2. Resting T cells do not express the high affinity receptor, but once activated, the anti-affinity receptor is rapidly expressed. The interaction between IL-2 and its induced cellular receptors triggers an increase in the proliferation of effector T cells required for full expression of the immune response. IL-2
Taking the / IL-2r complex as an example, in many of the diseases described herein, this physiological regulation is disrupted (primarily by neoplastic transformation and secondarily by viruses). Or self-sustained (autoimmune response / disease, transplant rejection), leading to systemic multiple organ failure.

Further information on cytokines and their receptors can be found in Callard RE, Geari
ng AJH, the Cytokine-Factbook, Academic Press-Harcourt Brace & Company
, Publishers, 1994, 18-25.

Accordingly, high affinity receptors include those having an affinity constant of 10 ⁻¹⁰ M or less, and more specifically, those having an affinity constant of 10 ⁻¹¹ M or less. Examples of high affinity receptors include those having an affinity constant between 10 ⁻¹¹ and 10 ⁻¹² M. For example, for interleukin 2 (IL-2), its IL
Based on the affinity for 2, it can be divided into three types of receptors with an affinity constant of 10 ⁻¹¹ M (high affinity), 10 ⁻⁹ M (medium affinity) and 10 ⁻⁸ M (low affinity). (Reference materials 1-4). The IL-2 receptor is well described in the prior art literature (Refs. 5 and 6).

[0011] TNF-α has been reported to have two high affinity isoform receptors on TNF target cells. These target cells are macrophages and osteoclasts (
Reference 7). M-CSF (macrophage colony stimulating factor) has a high affinity receptor on macrophages and osteoclasts. The high affinity receptor is a 150 Kda glycoprotein (Reference 8).

[0012] High affinity receptors have also been reported for IFN (interferon). IFN-γ has a 90 KDa glycoprotein as a high affinity receptor. Various receptors present on activated lymphocytes, macrophages, endothelial cells, and fibroblasts are recognized as high-affinity receptors for IFN-α and IFN-β (Reference 9).

In the case of FGF (fibroblast growth factor), activated fibroblasts, macrophages,
Endothelial cells, chondrocytes, astrocytic cells, glioma cells, hepatocytes, epithelial cells, nerve cells, ovary cells, pituitary cells, mesodermal and neuroectodermal lineage cells such as keratinocytes, 140 KDa glycoprotein There is a high affinity receptor that is The pharmacological properties of FGF are primarily angiogenesis, ovarian steroidogenesis,
Osteoblast activation and (fetal) nerve growth (Ref. 10).

[0014] IGF (insulin-like growth factor) has a high affinity receptor on an etrotetrameric complex present in various tissues and mammalian adenocarcinoma (Ref. 11).

[0015] Transforming growth factor β (TGFβ) is similar to IFG. TGFβ is a non-glycosylated homodimer protein secreted by fibroblasts, epithelial cells, platelets, astrocytic cells, monocytes, osteocytes and glioblastoma cells. Physiological target cells include primary fibroblasts, osteoblasts, neutrophils, hematopoietic progenitors,
T / B lymphocytes and various tumor cells. Cytokines interact with high-affinity receptors expressed by target cells in a paracrine microenvironmental stimulus response located on the cell surface of superior organ cells. These are type 1 or type 2 receptors (
55 and 80 Kda) and can bind to TGFβ1, 2, and 3.

GM-CSF (granule cell / macrophage colony stimulating factor) and SCF (stem cell factor) have a dimeric high affinity receptor in pluripotent cells in bone marrow (Reference Material 12). . G-CSF (granule cell colony stimulating factor) is also
It has a high affinity receptor but is present only in pluripotent cells in bone marrow.

EPO (erythropoietin) has multimeric high affinity receptors on erythroid progenitor cells in the bone marrow.

[0018] IL-6 (interleukin 6) has an α- / β-high affinity receptor. The α-chain binds to IL-6 with low affinity and exists in a soluble form. The β chain is 130K
It is a protein of Da and binds simultaneously with IL-6 / IL-6r to form a trimeric complex and initiates target cell stimulation. Induction of the IL-6 high affinity receptor following specific stimulation occurs primarily on activated cells such as T / B lymphocytes, fibroblasts, bone marrow progenitor cells, nerve cells, epidermal cells and stem cells. . In addition, multiple myeloid cells produce IL-6, express the IL-6 receptor, which acts as an autocrine cancer growth factor, and simultaneously induce osteoclastogenesis (osteolytic disease). IL-6 derived from stromal cells is also involved in bone metastatic disease through various tumor histological types.

Several studies (in vitro, animal models, and humans) have been performed using radiolabeled interleukin-2. Recombinant of the protein was prepared by the chloramine T method (preclinical test) or the lactate peroxidase method (clinical test).
Labeled with 3. The iodine-labeled cytokine was purified using the RT-HPLC method, and the radiolabeled cytokine was separated from the non-radiolabeled cytokine.

Radiolabeled studies in which naturally activated lymphocytes are injected intravenously (iv) into pathological conditions that are a major component of the pathogenesis of the disease have been performed, and in vivo ultra-low dose radiolabeled IL-2 The distribution was examined (reference materials 13 and 14). The models tested were autoimmune diabetes (diabetic rats), patients newly diagnosed with autoimmune type 1 diabetes, patients newly diagnosed with autoimmune thyroiditis, and chronic inflammatory bowel disease (pediatric steatosis). And Crohn's disease) (Reference materials 14, 15, 16).

The results of these studies are as follows. 1. In vivo administration of very low doses of labeled IL-2 was feasible in animals and humans without side effects. 2. After the distribution phase, labeled IL-2 was detected and retained only when the target site was the target of the autoimmune pathological condition in question, but in normal organs (ie, organs that were not attacked by the immune system). Was not detected and retained. 3. Labeled IL-2 maintains its ability to bind to IL-2 receptor on activated lymphocytes in vitro.

Other in vitro studies on lymphocytes have shown that following binding between IL-2 and its renewable receptor, the entire complex (IL-2 / IL-2 receptor) is rapidly reduced. It clearly shows that it is internalized (uptake) in the cytoplasm of lymph cells. This process is rapid (5-10 minutes) and occurs only when the isoform receptor has the highest affinity for IL-2. After this uptake, the endosomes in the cell compartment become progressively acidic (the pH drops to 5 through the action of multiple proteins on the endosome membrane, which acts as a proton pump), and two molecules (IL-2 and its receptor) ) Are divided and exert their effects separately (Reference Material 1).

To selectively target cells with high affinity IL-2 receptors, I
A fusion protein of the L-2 sequence and diphtheria toxin has been utilized. As the method, a method of constructing a diphtheria toxin-related IL-2 fusion gene encoding a recombinant toxin in which the diphtheria toxin receptor binding domain has been substituted with amino acids 2 to 133 of IL-2 has been adopted. This chimeric IL-2 toxin is E. coli K-12
It has been shown to selectively inhibit its protein synthesis only in a T cell line expressed in a recombinant strain of and expressing the high affinity IL-2 receptor (Ref. 22).
Similarly, chimeric proteins comprising sequences of Pseudomonas exotoxin (PE) and IL-2 ("IL-2-PE40") can provide a lymphokine-mediated method of providing toxins to IL-2r expressing cells ( Reference 6).

The chimeric IL-2 diphtheria toxin does not contain a biologically active IL-2 sequence,
Contains only the receptor binding sequence. This protein has entered a Phase III trial for the treatment of T-cell lymphoma (scrip, January 13, 1995 and New Scien
tist, November 1, 1997). Since blastic leukemia cells usually retain only low / medium affinity isoform IL-2 receptors (although only a small portion of the total receptors have high affinity receptors), IL- Includes intravenous administration of high doses of the protein to destroy neoplastic cells with two receptors. Such treatments often exhibit high toxicity. Furthermore, as chimeric proteins, the compounds have immunogenicity.

[0025] Since IL-2-PE40 is thought to have only limited activity on human T cells, other molecules have been constructed in studies of more active drugs.

WO 92/20364 is a first protein (particularly a cytotoxin) that is a molecule that can reduce cell viability, and a second protein that can specifically bind to cytokine receptors (particularly all or binding sites of cytokines). A hybrid molecule comprising two proteins is described. The second molecule targets a cytokine receptor to the first molecule, and is exemplified by IL-2. Since the molecule blocks the growth of target cells, it is preferred that the IL-2 moiety is deficient in IL-2 activity.

[0027] The present invention relates to the perspective of predicting that a medicament containing a proliferative activity promoter, such as active IL-2, can be beneficially used to provide a pharmaceutically desirable effect to cells where proliferation is not desired. It is based on. For example, the medicament of the present invention controls or inhibits growth by utilizing a molecule containing an active growth promoter. The preferred embodiment is based on applications that use the high affinity receptor superfamily and can, for example, induce drugs or genetic material into specific cell lineages that are primarily involved in many clinical cases.

Accordingly, one aspect of the present invention resides in a composition (product, eg, compound) comprising a proliferatively active ingredient bound to a biologically active drug, wherein the composition comprises a cell in which the drug is growing. A composition that acts preferentially or selectively is provided. In a preferred aspect, there is provided a biologically active agent conjugated to a cytokine or growth factor having a high affinity receptor, or a molecule functionally equivalent to the cytokine or growth factor. Binding between the two domains of the product (biologically active and proliferatively active agent /
Cytokines / growth factors) can usually be destroyed under intracellular conditions, for example by hydrolysis.

Unlike other vectors that have been evaluated or are currently being evaluated, the proliferative active component (or, in a preferred aspect, a cytokine or growth factor) retains its functional activity,
The product can target the receptor. The proliferative active ingredient binds to the receptor and is subsequently taken up by the cell, so that each active domain of the product (proliferative active ingredient and biologically active agent) can perform its function. In commercial products, the two domains of the product are expected to be separated within the cell, but this is not absolute and the invention is not limited to products that are cleavable inside the cell. In a preferred embodiment, the events following binding of the proliferative active ingredient to the receptor include incorporation of the product (typically a fusion compound) into the cytoplasm (through the endosome pathway), acidification of the endosome (proton H + pump Mechanism), and the separation of the functional domain into the receptor domain, the growth active domain and the active action element domain. Since the receptor domain and the growth activity domain (generally, a cytokine or growth factor domain) completely retain their functions, the growth activity domain (
Cytokines or growth factors) initiate cell activation / division via DNA interactions (G2-M phase condensation).

The present invention relates to a method for producing a proliferating active ingredient, which is usually formed by a covalent bond cleavable in a cell.
That is, it provides, but is not limited to, a product to which a component that causes cell proliferation is bound. In a preferred embodiment, the proliferative active ingredient has a high affinity receptor, and a very low dose such that only targeted cells express the high affinity receptor for the proliferative active ingredient (usually a cytokine or growth factor). The product can be administered at As already mentioned, the product is taken up into the target cells, where the biologically active ingredient usually separates from the proliferating active ingredient.

[0031] In yet another aspect, the present invention is a method of preventing or treating a disease or disorder involving cells having high affinity receptors for cytokines or growth factors, particularly when located in said cells. A method comprising administering to a patient an effective amount of a product that is therapeutically active and comprises the cytokine or the agent conjugated to the growth factor. The products and preparations containing them form another aspect of the present invention.

The present invention further relates to the use of pharmacologically, in particular, cells having a high affinity receptor for proliferatively active agents, cytokines or growth factors, for the purpose of incorporating biologically active agents into cells. Of the present invention.

Another aspect of the invention is the incorporation of a biologically active agent into cells that have a high affinity receptor for the product's proliferatively active agent, cytokine or growth factor, and optionally lymphatic. Use of a product of the invention for the manufacture of a medicament for stimulating cell proliferation.

Biologically active agents are substances which, in one preferred class of products, are effective in killing or inactivating proliferating cells, including physiologically active agents and proliferative active agents. Activities complement their activities. Such biologically active agents are antiproliferative agents, such as antiblast agents or chemotherapeutic agents that interfere with DNA replication. One class of proliferatively active agents of the product promotes lymphocyte replication. For example, IL-2 and antisense DNA / RNA constructs designed to block the retrovirus gene have the following effects.

A. In infected cells, stimulation of replication by IL-2 also stimulates replication of the viral genome, resulting in stronger antisense DNA / RNA inhibitory activity. b. Immunostimulation of uninfected lymphocytes with the potential for increased immune surveillance

The features of the preferred embodiment include: The product is a combination of two existing components (or components functionally equivalent thereto), each retaining its function and optionally maintaining its entire structure (with another component ). -The product can be produced, for example, by chemically combining the two components using standard techniques. -The product can be administered in very low doses, so there is little or no systemic toxicity. -Growth factors / cytokines stimulate the immune system and active ingredients induce therapeutic effects. -Biodistribution can be estimated and good-Good targeting-Low immunogenicity

A feature of the first aspect of the invention is that the biologically active agent (usually a pharmaceutical) is bound to the proliferative active ingredient. Thus, unlike conventional chimeric proteins containing only the receptor binding domain of IL-2, these products induce cell proliferation and anti-proliferation even at very low concentrations in the case of proliferating active ingredients with high affinity receptors. The agent can be highly activated. Therefore, the present invention can reduce systemic toxicity. Another advantage, at least for IL-2, is that IL-2 induces expression of the high affinity IL-2 receptor when the relevant antigen is present.

Hereinafter, the present invention will be described by way of examples with reference to specific specific cytokines, growth factors, biologically active agents and diseases. Of course, the present invention is not limited to these particular features.

The cytokine is an interleukin, such as TNF, for example M-CSF,
IFN, for example, FGF, IFG, TGF, for example, GM-CSF, S
CF, G-GSF or EPO. The cytokine is preferably a human cytokine.

[0040] The growth factor is a hematopoietic or lymphopoietic growth factor. These are a family of glycoprotein hormones that regulate the survival, proliferation and differentiation of progenitor cells, in addition to affecting the functional activity of mature lymphohematopoietic cells.

Preferred growth factors include: Erythropoietin (Epo), GM-CSF, G-CSF, SCF (stem cell factor), multi-CSF (also known as interleukin-3), M-CSF, E-CSF (or interleukin-5), IGF-1 (insulin-like growth factor), PDGF (platelet-derived growth factor), TGFβ2 (transforming growth factor-β2).

The cytokine or growth factor (or proliferative agent) may be naturally occurring or a mutein, which is a natural molecule modified by one or more amino acid changes (deletion, addition or substitution). Such muteins that can be used in the present invention have the biological activity of the native protein and have a functional affinity for the receptor (and, in one class of embodiments, a functional affinity for the high affinity receptor). ) And, together with the receptor, the ability to form products that are taken up by receptor presenting cells.

The cytokines and growth factors are preferably recombinant molecules, but can also be produced by culturing cytokine or growth factor producing cell lines, such as peripheral blood lymphocytes.

In one class of embodiments, the product confers a high affinity to cytokine or growth factor high affinity receptors and is a molecule that is functional to form a complex with these receptors for uptake by receptor presenting cells. And a biologically active compound bound to. In a particular class of products, the molecule is a natural or mutated cytokine, or a fragment thereof. In another product class, the molecule is a natural or mutant growth factor, or a fragment thereof.

A particularly preferred cytokine is interleukin-2 (IL-2). IL
-2 is a lymphokine produced by normal peripheral blood lymphocytes, and induces proliferation of T cells stimulated with antigens or mitogens exposed to plant lectins, antigens or other stimuli. IL-2 is available from Morgan, DA, et al., Scinece (197
6), 193: 1007-1008. It was subsequently called T cell growth factor because it could induce the proliferation of stimulated T lymphocytes, but now it has the ability to regulate various functions of the immune system in vitro and in vivo in addition to its growth factor properties. It has been identified and has been renamed Interleukin-2 (IL-2).

Interleukin-2 can be produced by culturing human peripheral blood lymphocytes (PBL), for example, as described in US Pat. No. 4,401,756. As a preferred alternative, IL-2 may be recombinant. Tanigchi, et al., Nature (1983
), 302: 305-310 and Devos, R., Nucleic Acid Research (1983), 11: 4307-4323.
Report cloning and expression in a microorganism of the human IL-2 gene.

US Pat. No. 4,518,584 discloses a variant of IL-2 in which the 125th position normally occupied by the cytosine of the wild-type or natural molecule is replaced by a neutral amino acid such as serine or alanine. It is described and claimed. Oxidation-resistant variants of IL-2 that are biologically active are prepared by substituting each methionine residue in the protein from which the mutant protein is obtained with a conservative amino acid such as alanine. Methionine residues are sensitive to the oxidation of chloramine T or peroxidase. These I
The L-2 mutein retains the biological activity of native IL-2. U.S. Patent Nos. 4,530,787 and 4,569,790 disclose recombinant natural IL-2.
And a method for producing a mutant protein thereof, and a form for producing IL-2, are claimed. The US patent is incorporated herein by reference in its entirety.

The IL-2 mutein desala ₁ -IL-2ser ¹²⁵ has the trade name Prol
Enkin by Chiron B.S. in Amsterdam, The Netherlands. V. More commercially available.

In the broadest aspect of the invention, in principle the biologically active agent is any compound that effectively affects the function of the cell into which the active agent is taken up. However, it is usually a species that acts selectively or preferentially on growing cells and in such embodiments is not a polypeptide cytotoxin such as, for example, Pseudomonas exotoxin or diphtheria toxin. In a preferred embodiment, the active agent is an immunosuppressant,
For example, known immunosuppressants. In one embodiment, the active compound comprises a known sequence, such as an antisense nucleotide sequence, eg, an anti-oncogene sequence. In another embodiment, the active agent is an enzyme inhibitor, for example, a viral reverse transcription inhibitor. Alternatively, the active agent is an anti-cancer or other anti-cancer agent. In a slightly preferred embodiment, the active agent comprises a radioisotope.

Particularly preferred are products comprising a biologically active agent conjugated to a cytokine or growth factor or a molecule functionally equivalent thereto, said biologically active agent being an antiproliferative agent, a nucleotide. It is selected from the group comprising compounds that interfere with synthesis, radioisotopes, gene and antisense nucleotide sequences, and cytokines or growth factors with target cells capable of presenting high affinity receptors thereto. The biologically active agent is preferably an immunosuppressant, an anti-slow inhibitor or an anti-cancer agent.

These particularly preferred products are those wherein the biologically active agent is cyclosporine, F
KK506, thalidomide, dihydrofolate reductase inhibitor, antiblast agent, platinum coordination complex, vinca alkaloid, purine analogs, pyrimidine analogs, corticosteroids, viral reverse transcriptase inhibitors or antisense nucleotide sequences. A specific classification of the product is that if the biologically active agent is cyclosporine, vinca alkaloid,
FKK506, thalidomide, methotrexate, azathioprine, cyclophosphamide, actinomycin D, daunone bicine, doxone bicine, bleomycin, rhenium radioisotope, yttrium radioisotope, 3′-azido-3′deoxythymidine, binds to nucleotide sequence of virus It is an antisense nucleotide sequence or an anti-oncogene nucleotide sequence.

[0052] In some embodiments, the active agent is not a prodrug. Of course, prodrugs are also included in the present invention, provided that the metabolism required to convert the prodrug to the active agent is localized in the cytoplasm of the target cell.

Preferably, the products of the present invention act only on cells that present high affinity receptors for cytokines or growth factors, typically lymphocytes or other cells involved in the immune response. The effect of the products of the invention on target cells depends on the function of the active agent.

[0054] The biologically active agent is preferably not a polypeptide. Preferably, it is a monomeric organic compound. The biologically active agent may include a metal.

When the biological agent is an organic compound, the active agent is usually covalently linked to a proliferative active ingredient, especially a cytokine or growth factor. That is, the products of the present invention are often molecules. The metal-containing product is usually an organometallic complex that includes an organic moiety covalently linked to the growth active.

The molecular ratio of the active agent: proliferative active ingredient in the products of the present invention is not critical. That is, while the present invention includes a ratio of 1: 1 or less, in some embodiments, the ratio is 1: 1.
More than 1: 1, for example 1: 1000 or more, in other words, more than one active drug molecule / atom can be attached to each proliferating active ingredient.

Hereinafter, the products of the present invention will be described in more detail by way of examples with reference to exemplary products and product classifications.

Interleukin-2 / cyclosporin fusion product Cyclosporin, a second-generation immunosuppressant, has an extremely high ability to selectively inhibit the activity of T cells (Ref. 17).

Unlike first generation immunosuppressive drugs such as azathioprine, methotrexate and cyclophosphamide, therapeutic concentrations of cyclosporine do not cause myelotoxicity. Cyclosporin inhibits the initial cellular response to antigenic and regulatory stimuli, primarily targeting T lymphocytes.

Cyclosporine rapidly and significantly inhibits IL-2 production by T cells and generally reduces the production and release of other lymphokines in response to antigenic stimulation. At high intracellular concentrations, cyclosporin inhibits the expression of receptors for IL-2.

Thus, cyclosporin and especially cyclosporin-A (Cs-A) are widely used for maintaining kidney, liver and heart transplants. The use of Cs-A for autoimmune diseases is uncommon due to side effects (acute or delayed) associated with the therapeutic range used during systemic administration and the duration of the treatment.

The major clinical toxicity of cyclosporine is nephrotoxicity, usually 25% to 7% of treated patients.
Occurs in 5%. This toxicity is dose-related and irreversible in some patients.
Reduction of cyclosporinemia within the therapeutic window leads to exacerbation of the underlying disease (transplant rejection). Other systemic side effects include increased blood pressure due to hypertension, neurotoxicity, and systemic immunosuppression. This systemic reduction in immunity also increases the incidence of malignancy in transplant patients.

In the present invention, a suitable pharmaceutical product comprising chemically bound IL-2 and cyclosporin (Cs-A or an analog of the same drug class) provides very specific immunosuppression. That is, a novel third generation immunosuppression method is provided. The system retains the immunosuppressive pharmacological properties of cyclosporine on lymphocytes, but its effects are naturally activated during the inflammatory process (graft rejection, autoimmune diseases) and the complex (
It is selectively directed only to lymphocytes that can take up IL-2 / Cs) (lymphocytes that retain high affinity receptors for IL-2).

A very low dose of IL-2 / Cs is administered intravenously (iv) or subcutaneously (sc) and
The active compound can be targeted in vivo. This means that extremely low doses of cyclosporin are available, as in fact all drugs are trapped by activated lymphocytes. Thus, while maintaining the pharmacological properties of the active compound (the intracellular concentration of the immunosuppressant in lymphocytes is very high), there is no systemic toxicity (I
The plasma concentration of L-2 / Cs is very low). From this immunological point of view, it is possible for the first time to achieve true oligoclonal immunosuppression.

Cyclosporine could be substituted for structurally related or non-structurally related compounds with a similar mechanism of action, such as thalidomide or FK506.

The interleukin-2 / methotrexate product methotrexate, and other dihydrofolate reductase inhibitors, a class of antineoplastic agents, are useful in the treatment of certain types of autoimmune and inflammatory diseases, as well as in cyclosporine. Has been used to prevent graft-versus-host disease in bone marrow transplantation (Ref. 17).

The immunosuppressive activity of methotrexate is achieved by inhibiting T cell (and possibly B cell) replication and functional activity as a result of its relatively selective effect on DNA synthesis. The agent can also be used to treat severe active rheumatoid arthritis and severe psoriasis in adults.

The clinical toxicity of methotrexate is strong and the standard toxicity of chemotherapy: severe bone marrow toxicity,
It shows mucositis, liver fibrosis, sclerosis, and pulmonary toxicity.

Long-term administration of methotoxerate, like other antineoplastic agents, increases the incidence of infections, malignancies, and sterility in patients. The IL-2 / methotrexate complex is
Acts in the same manner as the IL-2 / cyclosporin complex. IL-2 puts methotrexate in activated lymphocytes involved in the autoimmune condition or immune rejection in question. These are the only cells in the body that have a high affinity IL-2 receptor on the membrane. Methotrexate inactivates only these T lymphocytes by blocking intracellular DNA synthesis via inhibition of intracellular dihydrofolate reductase (
Oligoclonal immunosuppression). The remaining immune cells, and cells that do not experience myelotoxicity, all maintain their reactivity. This allows the patient to respond to the infectious stimulus. Low dose of IL-2 means low dose of methotoxide. Thus, the pharmacological effects of methotrexate are retained in the targeted lymphocytes by the system, but the plasma concentration of whole complex, or free methotrexate, is virtually undetectable.

Intracellular nucleophiles, such as the interleukin-2 / azathioprine product glutathione, cleave the prodrug azathioprine to mercaptopurine. Subsequently, the purine analog is converted to a mercaptopurine-containing nucleotide and acts on the synthesis and utilization of RNA and DNA precursors (Ref. 17).

Azathioprine is one of the most important first generation immunosuppressants (in combination with prednisolone) for transplant maintenance. It is also used in the treatment of some autoimmune diseases, such as severe and recurrent rheumatoid arthritis.

Unfortunately, this potent antiblastic compound has a systemically distributed consequence (ie,
Consequences of lack of specificity for immune cells), myelotoxicity (leukopenia and thrombocytopenia), nausea, vomiting, hepatic vascular obstruction, and germline toxicity (amenorrhea and hypo-azoospermia) Shows strong toxicity including

When a low dose of the IL-2 / azathioprine conjugate molecule is administered (parenterally), the prodrug azathioprine only enters activated lymphocytes that have a high affinity receptor for the cytokine IL-2 . These cells (primarily of the T cell line) are the main cells involved in the immune attack in certain immune diseases (transplant rejection or autoimmune diseases). Only in these cells, the activating compound mercaptopurine reaches pharmacological concentrations and can inhibit cells by oligoclonal immunosuppression.

The high specificity of this therapy greatly reduces the total dose of antiblast compound administered in vivo, and greatly reduces or eliminates drug-induced toxicity.

Interleukin-2 / anti-tumor antibiotics (actinomycin D, daunorubicin , doxorubicin, bleomycin and all related compounds) This therapeutic complex has many potent uses widely used in the treatment of many malignancies. Antitumor agents (antibiotics).

Intracellular mechanisms of action include interactions with DNA and RNA at multiple molecular levels where intracellular concentrations exceed natural DNA repair and cell recovery mechanisms and result in cell death (reference material). 17).

[0077] The patterns of clinical toxicity are extremely diverse and severe. All normally replicating cells in the body will be damaged by the lack of these properties. These compounds circulate in the body after administration and interact with most cells.

When parenterally administered very low concentrations of the IL-2 / antitumor antibiotic conjugate, all active compounds (antitumor antibiotics) are released into the body as a result of autoimmune disease or during transplant rejection. Exclusively captured by existing activated lymphocytes. The intracellular concentration of the active compound in target cells (primarily activated T cells) reaches a level of pharmacological action that can inhibit or kill only those cells. Very low levels of conjugates in plasma, free IL-2 or free antitumor antibiotics, are diluted by a large volume distribution so that they have no significant effect on off-target cells.

Platinum coordination complexes, vinca alkaloids, purine analogs, pyrimidine analogs, and corticosteroids can form complexes with IL-2 in a manner similar to the active agents. Representative vinca alkaloids include vindesine, vinorelbine and vinleurosine.

Interleukin 2 / radioactive isotope product (rhenium, yttrium) In these complexes, the active agent bound to IL-2 is a radioactive cytotoxic isotope. Iodine-labeled IL-2 has been used for diagnostic purposes, but cytotoxic isotopes that bind to IL-2 (microscopic high-energy radiation) to provide a radiation field within target cells (T cells) Isotope that kills cells using a field). These in vivo specific radioimmunotherapy treatments could be used to target immune attacks in the microinflammatory environment during the process of autoimmune disease or during rejection of transplanted organs.

Interleukin-2 / AZT Product In this example, the primary target is human immunodeficiency virus (HIV) infection. AZT (zidovudine, 3'-azido-3'-deoxythymidine) is an active agent against HIV-1 retroviruses, like other congener antiviral compounds that have been experimentally evaluated for HIV infection.

AZT is phosphorylated by an enzyme in the cell to each deoxynucleoside triphosphate which inhibits viral reverse transcriptase. This antiviral selectivity is due to its greater affinity for reverse transcriptase than for human DNA polymerase (Ref. 16).

This reduces the replication of the HIV virus in CD4 lymphocytes, a special subpopulation of lymphocytes that are major targets of the HIV retrovirus, as well as AIDS
It is widely used in HIV-infected patients for the purpose of delaying the onset of systemic immunodeficiency that progresses to disease.

During prolonged treatment, the concentration of the drug in the body must be maintained at a high level in order to prevent viral replication as much as possible. This allows H
AZT also penetrates into cells where IV is not naturally present, causing moderate to severe side effects. This side effect is mainly due to myelotoxicity and immunosuppression.

With interleukin-2 / AZT (or other anti-HIV compounds), the active drug is trapped and taken up only by lymphocytes with a high affinity receptor for IL-2, so the volume of distribution and other cells Interaction with the active antiretroviral compound is reduced.

In this special case, ie, HIV infection, the rational use of the complex is further enhanced by the specific state of the disease. The cleavage of the complex intracellularly by IL-2 (an active compound that promotes lymphocyte replication) and an antiretroviral agent (an active compound that blocks viral replication) provides the basis for a synergistic reduction. (A) having the benefit of enhancing the general host immune surveillance of patients in an immunosuppressed state;
Immunostimulation of lymphocytes that have not been infected with HIV (active antiretroviral compounds accumulated inside the cells are dangerous for the HIV genome but inactive for cell biochemistry); and (b) HIV In infected lymphocytes, the replication stimulus provided by IL-2 promotes the replication stimulus for the HIV genome, resulting in HI
It increases the antiretroviral activity of an anti-retroviral agent coexisting in V-infected lymphocytes.

Interleukin-2 / antisense products IL-2 / antisense fusion products are useful for introducing specific antisense sequences (oligonucleotides) into lymphocytes bearing IL-2 receptors. .

An antisense oligonucleotide is a short synthetic short nucleotide that contains a sequence that is complementary to a target mRNA or DNA. Specific DNA associated with the HIV genome
Alternatively, antisense compounds that inhibit RNA sequences can be made. However, conventionally, there is no known method for introducing such a special sequence in vivo. Utilizing a cytokine (eg, IL-2) incorporated into a target cell, an antisense compound against a specific site in the HIV genome (eg, a genome encoding an envelope protein, or a genome for the enzyme transcriptase) can be synthesized using, for example, I.
It can be specifically introduced into the patient's lymph cells using a pharmaceutical method such as V administration.

The IL-2 / antisense product is affected by the transformation of the neoplasm, and the antisense compound or anti-oncogene is introduced into a T cell line known to have a genetic mutation or up-regulation of an oncogene. Can also be used for introduction.

Therapeutic Diseases The IL-2 / active compound fusion products are useful for diseases in which lymphocytes are primarily involved in tissue damage and progression of the substantial disease in question.

In summary, immunosuppressive therapy via the interleukin-2 high-affinity receptor acts pharmacologically only on recently activated lymphocytes, whose structure is on the cell membrane. No activation signals are present on resting T cell surfaces and other non-lymphoid tissues. Thus, very low doses of cytokines / immunosuppressants can be targeted.

Receptors are activated because they are only transiently expressed during the short growth phase in which lymphocytes respond to antigenic stimuli (autoantigens in autoimmune diseases and heterologous antigens at the time of transplantation). Selective immunosuppression in vivo (oligoclonal immunosuppression) that targets only lymphocytes. The pharmacological effects are completely different from the general immunosuppressive effects exhibited by ordinary immunosuppressants.

Diseases that can benefit from this method include autoimmune disease, transplant rejection, H
IV infections and lymphoproliferative disorders.

Autoimmune Diseases Autoimmune diseases are a widespread pathogenesis involving a common etiological pathway, namely immune attack on target organs due to abnormal recognition of tissue and / or cellular antigens by the immune system, especially T lymphocytes. It is a disease.

This immune attack is driven by T cell-mediated cytotoxicity, humoral autoimmune antibodies produced by B lymphocytes, a network of complement activation and consumption, and eventual tissue damage. It is well known that abnormal activation of the T lymphocyte line plays a central role in all autoimmune diseases.

[0096] Such clinical diseases and target organs in such cases include the following. 1. 1. Autoimmune diabetes (type 1 diabetes) → pancreas of endocrine glands 2. Autoimmune thyroiditis (Hashimoto's disease and others) → thyroid gland 3. Autoimmune hepatitis (chronic active hepatitis) → liver 4. Rheumatoid arthritis → synovium / joint / visceral organ 5. Autoimmune nephritis (glomerulonephritis) → kidney 6. Uveitis (Behcet's syndrome) → eye 7. Multiple sclerosis → CNS / PNS 8. Sjogren's syndrome → salivary gland Scleroderma → skin / visceral organs 10. Polydermatomyositis → skin / muscle / visceral organs 11. 11. Systemic lupus erythematosus (SLE) → internal organs / skin / hematopoietic organ / mucosa 12. autoimmune hemolytic anemia → red blood cells 13. Sudden thrombocytopenic purpura (TTP) → platelets 14. Autoimmune neutropenia → neutrophils Vasculitis → blood vessels 16. Crohn's disease → bowel 17. Ulcerative enteritis → intestine Pediatric steatosis → bowel Psoriasis → skin / joint / visceral organ 20. 21. Sarcoidosis → lung / visceral / skin Atopic syndrome

[0097] For many of these pathological conditions, pathogenic lymphocyte-mediated reactions and cytotoxicity are noted.

Evidence supporting the role of T cells in the pathogenesis of certain diseases and in the progression of target tissue damage is clear. In many of the above diseases, CD4 cells (a cytotoxic subset of T cells) are the predominant T cell phenotype in target tissues. T cells express multiple activation markers. In experiments, there is evidence that T-cell targeted interventions such as thoracic duct drainage, total lymph node irradiation, and cyclosporine administration improve autoimmune disease. In addition, AIDS patients with reduced CD4 cells generally have a lower degree of active autoimmune disease.

[0099] Most autoimmune diseases are treated by utilizing immunosuppressive and anti-inflammatory drugs to reduce the function of the immune system. Because this treatment strategy is implemented non-specifically,
At the same time, iatrogenic toxicity also occurs, resulting in failure to manage the progress of the disease as a whole.

All lymphocytes involved in the acute phase of this autoimmune attack have high-affinity IL-2 receptors on the membrane. These are antigen activated or cytokine activated lymphocytes with high affinity for IL-2.

Parenteral administration of very low doses of IL-2 / immunosuppressive product can be used as a vector of pharmacologically active drugs to effect IL-2 immunosuppression. IL-2 / immunosuppressive products selectively bind only to cells with a high affinity receptor for IL-2,
And interact. This means that immune cells involved in tissue damage and disease progression are selectively inhibited, strongly healing patients with chronic diseases, or reducing their relapse rate.

Utilizing the novel invention, very specific immunosuppression (referred to as oligoclonal deficiency), maximizing the effects of immunosuppressive drugs and counteracting many toxicities.

When IL-2 is used at low or very low doses, it has always been shown to be completely safe and can be administered for long periods of time. There is no effect (or toxicity) when only immunosuppressants are used at very low concentrations. However, when the two are put together, the two compounds are at high concentrations where needed (intracellular).

Recombinant proteins such as recombinant IL-2 and other recombinant cytokines and growth factors usually have low immunogenicity and good tissue distribution. After parenteral administration, all tissue compartments within the body are exposed, including all lymphocytes (circulating lymphocytes, lymphocytes in tissues and lymph nodes in lymph nodes).

Ultra-low doses of immunosuppressive or anti-blast agents, such as cyclosporine, cytostatics, and any of the above mentioned drugs, may be any of the "normal" toxicities commonly experienced by patients (bone marrow toxicity,
Renal function, metabolic disorders, cerebral dysfunction, cardiovascular disorders, infections, opportunistic infections and malignancies).

[0106] acute or chronic rejection of transplanted organ transplant, associated with heterologous antigen presented to the host T cells (specific antigen to transplant donor organs). Following antigen presentation and recognition, immune cells enter a proliferative phase during which high affinity receptors for IL-2 are expressed. This leads to a cytotoxic process, in which the transplanted organ is damaged and subsequently fails.

The use of IL-2 as a targeting vector for immunosuppressants can extend the life of transplanted organs without the usual toxicity (acute, delayed and long term) associated with immunotherapy.

[0108] Allogeneic bone marrow transplantation (ABMT) is used for the treatment and treatment of leukemia (both lymphoid and myeloid), Mediterranean anemia, and solid cancer. The product of the present invention provides a graft-versus-host disease (Graft-host disease) reaction of the donor's immune system to host tissue antigens without damaging the entire immune system (of transplant origin).
Versus-Host-Disease (GVHD) has the ability to reduce the frequency of occurrence. As a result, GVHD mortality (currently over 40%) can be reduced, infectious complications can be reduced, and a positive anti-tumor effect against minimal residual disease (referred to as graft versus leukemia effect) can be obtained.

Administration of low-dose IL-2 / AZT fusion products (or IL-2 / antiretroviral analogs or IL-2 / HIV genome fraction-specific antisense) of HIV infection
In o, an immunostimulatory effect on HIV-negative lymphocytes having high affinity receptor is shown. Antiretroviral compounds are introduced into the cytoplasm of HIV infected lymphocytes (CD4 cells) and result in the selective destruction of infected cells without affecting stimulated normal reactive lymphocytes.

Lymphoid Proliferative Disorders (Lymphoblastic Leukemia and Lymphoma) The IL-2 / cytotoxic fusion complex can be used to convert neoplastic lymphoid cells expressing the high affinity IL-2 receptor into non-lymphoid cells. It can be selectively killed without inducing significant systemic toxicity to the system compartment.

Products containing growth factors or cytokines other than IL-2 can be used for the following treatments.

[0112] TNF-α has macrophages and osteoclasts as its target cells. T
The NF-α / blocking compound product can be used for insertion of a blocking compound (which blocks cell function and / or kills cells) into macrophages. Such products include, for example, advanced solid tumors in which macrophage hyperstimulation and activation are involved in cachexia and tumor progression, mononuclear-macrophage neoplasms (eg, histiocytosis), transplants It may provide important tools for multiple pathological conditions such as rejection and GVHD, autoimmunity, and neurodegenerative diseases (the extracellular domain TNF receptor is similar to the nerve growth factor receptor).

M-CSF (Macrophage Colony Stimulating Factor) can be used to form products containing blocking compounds that are useful as TNF-α / blocking compound products for treatment of the same class of conditions.

M-CSF is also involved in the proliferation of microglia in the CNS. Therefore,
Other possible applications include degenerative diseases of the CNS, such as Alzheimer's syndrome, and bone diseases.

The product IFN / active compound (IFN-α, -β or -γ) is used to modify the function of activated lymphocytes, macrophages, endothelial cells and fibroblasts, or for example for HIV infection (AIDS) ) And other pathological conditions such as fibroblast-related diseases such as scleroderma.

[0116] Possible applications of FGF products include the use as anti-angiogenic factors for solid tumors,
And its application in blocking the overactivation of fibroblasts in scleroderma. The present invention relates to cells of the type described herein above as having the high affinity receptor for FGF when activated, the FG being capable of acting as an antagonist.
An F product can be prepared.

[0117] BIFG / antiblast products can be used to treat breast cancer. CNS (neural glia)
Due to the presence of the high affinity receptor therein, the product could be applied to some degenerative neurological disorders.

TGFβ products have similar applications as FGF and IFG fusion products.

The GM-CSF / active compound fusion product could be used to selectively kill myeloid blast cells involved in myeloid leukemia. The G-CSF product has similar pharmacological activity as the GM-CSF product, but binds to another type of high affinity receptor present only on pluripotent stem cells in the bone marrow.

[0120] Epo fusion products may be used for diseases such as erythrocytosis and erythroleukemia.

The Epo / gene sequence fusion product whose DNA fraction is the normal gene for the hemoglobin β chain can be used for normal gene transfer into the erythroid lineage of patients suffering from β type Mediterranean anemia. In this hereditary disease, an abnormal gene encoding a non-functional hemoglobin β chain is present in erythroid progenitor cells in bone marrow. Selective normal gene insertion into the myeloid erythroid lineage via the erythropoietin vector is an example of true in vivo gene therapy and may treat patients with this disease. A similar application is devised for another hereditary hemoglobin disease, sickle cell anemia.

IL-2 / fusion product is multiple myeloma, hyperactivation of osteoclasts (metastasis to bone)
Could be used to block cells with IL-6 high affinity receptors associated with cancer-related bone disorders, and osteoporosis.

Turning now to a group of products containing growth factors (or functionally equivalent with respect to receptor affinity), the following fusion products and target diseases are possible (“CTX
"Means cytotoxin).

Target Related Diseases of Fusion Products and Cytotoxins Epo / CTX in erythroleukemia (Di Guglielmo syndrome, FA
In the B classification, it is also known as M6 leukemia). GM- in chronic myeloid leukemia (CML), acute myeloid leukemia (AML)
2. CSF / CTX 3. G-CSF / CTX in CML, AML AML, acute lymphoblastic leukemia (ALL) and CML exacerbation phase (blast acute phase)
4. rSCF / CTX in 5. IL-3 / CTX in ALL, CLL, CML and AML Acute monocytic leukemia (FAB M5a and M5b) and monomyelocytic leukemia (
6. M-CSF / CTX in FAB M4) 7. M-CSF / CTX in Wegner's disease, granulomatous disease, inflammatory breast cancer, giant cell vasculitis, sarcoidosis, histiocytic necrotizing lymphadenitis (Kikuchi disease) 8. IL-5 / CTX in eosinophil syndrome (Wegner, polymyositis, granuloma, systemic allergic skin reaction, parasitosis) GM-CSF (or) G-CSF in myelodysplastic syndrome (MDS)
9. (or) SCF (or) IL-3 / CTX 10. IGF-1 / CTX in breast cancer 11. TGF / CTX in malignant transformation of bone (osteogranulomas, chondrosarcoma, fibrosarcoma) and fibrodysplasia syndrome (scleroderma) TGF / CTX in anti-angiogenesis (adenocarcinoma)

[0125] The growth factor fusion product is utilized as an active vector in gene targeting. The following discussion considers the same growth factors and interleukin 2 as described above. Importantly, these vectors are not only active as gene transferers, but also cleave DNA strands in target cells and further promote DNA reconstitution. Therefore, they are ideal for gene integration both in vitro and in vivo. No other system has comparable biphasic activity and does not carry the risk of viruses (currently used as vectors). Suitable fusion products and target diseases include:

Related products targeted by fusion products and genes 1. Epo / Hbβ gene in Mediterranean anemia 2. Epo / SS wild-type gene in sickle cell anemia (or sickle cell disease) GM-CSF / ABL_BCR gene in chronic myeloid leukemia (CML), Philadelphia + (Ph +) and its exacerbation phase (blast acute phase). 4. G-CSF / ABL_BCR gene in chronic myeloid leukemia (CML), Philadelphia + (Ph +) and its exacerbation phase (blast acute phase). 5. SCF / A in chronic myeloid leukemia (CML), Philadelphia + (Ph +) and its exacerbation phase (blast acute phase) and AMLPh + (a rare subtype of AMLs)
BL_BCR gene. 6. 6. IGF-1 / p53 tumor suppressor gene in breast adenocarcinoma 7. IGF-1 / antisense HER-2 Neu in thoracic gland Inborn deficiencies (usually these are polygenic deficiencies,
IL-2 (or) IL in severe combined immunodeficiency disease, inheriting DiGeorge syndrome, Neserov syndrome, ataxia telangiectasia, X-linked gamma globulinemia
8. -3 (or) GM-CSF / functional gene 9. IL-2 / function genes in selective deficiency of T lymphocyte function, such as hereditary purine nucleoside kinase deficiency (PNP syndrome) M-CSF / functional gene in congenital macrophage enzymatic monogene deficiency commonly found in lysosomal storage diseases. Lysosomal storage disorders include monoenzyme deficiencies (beta-galactosidase, beta-glucocerebrosidase deficiency in Gauche disease, alpha fucosidase deficiency in fucose storage disease, ceramidase deficiency in Faber disease, and hexoamini in Ty-Sachs syndrome. The majority of lipid storage disorders characterized by Dase A deficiency), mucopolysaccharide and glycoprotein storage diseases. All of these severe congenital disorders develop during fetal, young, and adulthood.

Wild-type genes encoding functional enzymes that are selected based on MCSF / specific deficiency, are incorporated into macrophages, and are transcribed into proteins can complement non-functional proteins and repair defects in vivo .

The prepared active agents and cytokines or growth factors, or equivalents thereof, utilize polyfunctional (eg, bifunctional) linkers that react with the corresponding functional groups on the active agent and the peptide hormone (or equivalent). And are preferably combined. Where the active agent is a radioactive isotope, a linker chelator that forms a covalent bond with the cytokine or growth factor, or its equivalent, can be used. In some embodiments, the two components are joined by an intracellularly cleavable bond. In another embodiment, the binding is stable within the cell.

The preparation of products containing polypeptides conjugated to other components is well known, for example, as in the case of fusion proteins, and therefore does not require the skilled artisan to describe the preparation techniques. Generally, preferred linkers are polyfunctional, especially bifunctional compounds that can react with at least two types of polypeptide molecules.

One typical technique involves the use of an acid-cleaving reagent for binding between two polypeptides. Such acid-cleavable linker reagents based on the functionality of orthoesters, acetals and ketals have already been described (Ref. 18), and bifunctional compounds whose hydrolysis rate constant increases with decreasing pH. It is. Crosslinkers react with proteins via heterobifunctional groups (eg, maleimide or N-hydroxysuccinimide ester) or homobifunctional groups (eg, bismaleimide or bissuccinimidyl).

In particular, three types of crosslinking agents are used. 1. Disuccinimidyl suberate. It is a homobifunctional crosslinker and contains an N-hydroxysuccinimide ("NHS") ester reactive group that is reactive toward amino groups. The crosslinker reagent chains cannot be broken. 2. Ethylene glycobis [succinimidyl succinate]. This is also a homobifunctional crosslinker reagent and contains an NHS ester reactive group that is reactive to amino groups. This crosslinker chain is cleavable. 4. Succinimidyl 6- [3- (2-pyridylthio) -propionamide] hexanoate. It is a heterobifunctional crosslinker and contains pyridyldithio and NHS ester reactive groups that are reactive towards sulfidyl and amino groups. This crosslinker chain can be cleaved.

The product in which the proliferative active ingredient (particularly a cytokine or growth factor) and the biologically active agent are cleavable by an acid has the potential to release the active agent in free form by cleavage of the product in the endosome. With benefits derived from benefits. Free drugs may have more effective intracellular effects than conjugated drugs.

For example, in the case of a nucleotide (DNA or RNA) active agent for gene targeting, a cytokine or growth factor can be covalently linked to the nucleotide. The resulting product will be mixed with nucleotides to form a protein / DNA complex.

The literature describes methods for attaching many radioisotopes to proteins, typically using bifunctional linker-chelators. For example, readers may read the textbook "Laboratory Techniques in Biochemistry and Molecular Biology", Vol.
ume 19, edited by RH Burdon and PH van Knippenberg, published by Elsevier and "S
synthetic Polypeptides as Antigens ", MHV Ban Regenmortel et al, Else
See Chapter 3 ("Peptide-Carrier Conjugation") of the textbook published by Ver. (1988). These publications also contain guidance on the binding of peptides to substrates other than radioisotopes. Gestin et al.
The preparation of such a linker-chelating agent comprising 1,2-diaminocyclohexane-N, N, N ', N'-tetraacetic acid as liquid is described. Gest
in describes the preparation of five linker-chelating agents having the general formula:

[0135]

Embedded image

Wherein R =-(CH_Two)₆,-(CH_Two)_Two-O_TwoC- (CH_Two)_Two-CO_Two− (CH _Two )_Two-, -CH_Two-S-CH_Two-,-(CH_Two)_Two-S- (CH_Two)_Two-Or-(CH _Two )_Two-SS- (CH_Two)_TwoIt is.

Linker chelators react with cytokines or growth factors, or functional equivalents thereof, and are subsequently utilized to chelate radioisotopes.

A review by TJ McKearn (Ref. 20) discusses techniques for coupling a radioisotope to a protein, typically using a linker chelator. The technique discussed by MaKearn relates to radioimmunoassays, but is also applicable to the products of the present invention.

[0139] Quadri et al. (Ref. 21) describe the preparation of stable and labile bonds between polypeptides (antibodies) and chelators. The chelating agent used is DTPA (diethylenetriaminepentaacetic acid). For the purpose of synthesis, a derivative aminobenzyl-DTPA (ABDTPA) was prepared and E
TPA is attached to a suitable linker. As the unstable linker, ethylene glycol bis (succinimidyl succinate) (EGS) and disuccinimidyl tartaric acid (DST) were used. EGS or DST is mixed, for example, in equimolar ratio with ABDTPA in an anhydrous solvent such as DMSO. To the resulting linker-chelator is added a suitable polypeptide in an anhydrous solvent (eg, DMSO) to form a polypeptide-linker-chelator molecule. In another technique described by Quadri, EGS or DST in the above method is a relatively stable hydrocarbon linker, DS.
Exchanged for S (disuccinimidyl succinate). A third technique described by Quadri is to react ABDTPA with thiophosgene (80% CHCl ₂ ) and give isothiocyanate benzyl-DTPA (ITCB-DTPA).
make. ITCB-DTPA was reacted with the amino groups of the protein in an aqueous liquid.

Suitable linkers for linking cytokines and growth factors to biologically active agents are described by Pierce & Warner (UK) Limited, 44 Upper Northgate Street, Chester C
Available from H14EF, UK, the literature provides further information.

Administered IL-2 and other cytokines or growth factors were administered parenterally at 10.00%.
0-1.000.000 international unit dose can be administered, preferably by intravenous, intramuscular or subcutaneous injection (less than 1 μg to 0.1 mg of recombinant protein) to obtain very low plasma concentrations. For example, plasma IL-2 concentration is 50% of the IL-2 high affinity receptor isoform.
% Can be a value close to the dissociation constant (kD) of the concentration of IL-2 at which the concentration saturates.
Furthermore, there are generally no systemic side effects at this dose range.

The products of the present invention are actually formulated as human or veterinary pharmaceutical preparations containing pharmaceutically acceptable diluent carriers or additives.

The formulation will be in the form of a liquid or suspension. The formulations are suitable for parenteral (eg, iv or sc) administration, but do not preclude oral preparations.

Accordingly, it is believed that the present invention provides a biologically active agent conjugated to a peptide hormone having a high affinity receptor, or a moiety that is a molecule that is functionally equivalent to the peptide hormone with respect to the high affinity receptor. . Peptide hormones include cytokines and growth factors. Examples are the molecules described above, including domains functionally equivalent to growth factors (eg, all or part of certain natural growth factors) and cytotoxins.

As used herein, “comprising” does not have an exclusive meaning; in other words, the object of this verb does not consist solely of the object, but rather the purpose of the verb. It is meant that the term can include words and one or more additional elements. Conservative expressions are interpreted similarly.

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Invest. Dennatol. 94: 275-325 5. EP 319012 (Du Pont) Waldmann, T .; A. (1993) Immunol. Today 1
4: 264-269 7. Cerami and Beuter (1988) Immuno. Toda
y 9:28 8. Suzu et al., (1992) J. Mol. Biol. Chem. 267: 4345 9. Sato et al., (1992) Cancer Res. 52: 444 Gabbianelli et al. (1990) J. Am. Biol. Chem. 249
: 1252 11. Zumstein et al. (1987) J. Am. Biol. Chem. 262: 12
52 12. Bussolino et al. (1989) Nature 337: 471 13. Signal A et al. (1987) The Lancet, Vol II,
No. 8558 14. Signal A et al. (1992) Nuclear med. Comm.
13: 713-72215. Chianelli et al. Nuclear medicine Process
ed. EANM 1991: 143-146 Bellan-hallel A et al. Immunology
cal Methods 119: 127-133 17. Godoman & Gilman The Pharmacologica
l Basis of Therapeutics, Eightth Edit
on 1990 18. Neville DM et al., (1989) J. Mol. Biol. Chem. 264
: 14653-1466119. Gestin JF et al., (1993) Nucl. Med. Biol. 2
0: 755-762 20. McKearn TJ. , CANCER Supplement, Ju
ne 15, 1993, Vol 71, No. 12,4302-4313 21. Quadri SM et al. (1993) J. Mol. Nuc. Med. 34: 938
-945 22. Williams DP et al., (1987) Protein Engineering (Prote
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[Procedural Amendment] Submission of translation of Article 34 Amendment of the Patent Cooperation Treaty

[Submission date] October 21, 1999 (1999.10.21)

[Procedure amendment 1]

[Document name to be amended] Statement

[Correction target item name] Claims

[Correction method] Change

[Correction contents]

[Claims]

──────────────────────────────────────────────────続 き Continuation of front page (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE ), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GE, GH, GM, HU, ID, IL, IS, JP, KE, KG, KP, KR , KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZWF term (reference) 4C076 AA95 CC29 CC42 EE59 FF68 4C084 AA01 AA02 AA03 AA07 AA16 BA44 CA62 DA01 DB52 MA05 NA13

Claims (36)

[Claims] 1. A product comprising a proliferative active ingredient conjugated to a biologically active agent that acts preferentially or selectively on proliferating cells. 2. The product of claim 1, wherein the bond between the agent and the component is cleavable in a cell. 3. The product according to claim 2, wherein said bond is cleavable by acid hydrolysis. 4. The product according to claim 1, wherein the target cells of the proliferative active component have a high affinity receptor for the component. 5. The product of claim 4, wherein said proliferative active ingredient is a cytokine or growth factor, or a molecule functionally equivalent thereto. 6. The product according to claim 5, wherein the proliferative active ingredient is a cytokine or a molecule functionally equivalent to the cytokine. 7. The cytokine, wherein the cytokine is IL, TNF and M-CSF, IFN, F
The product according to claim 6, which is GF, IFG, TGF, GM-CSF, SCF, G-CSF or Epo. 8. IL is IL-2 or IL-6, TNF is TNF-α, IFN is INF-α, INF-β or INF-γ, and TGF is TGF
The product of claim 7, which is β. 9. The product of claim 5, wherein said proliferative active ingredient is a growth factor or a molecule functionally equivalent to a growth factor. 10. The growth factor is erythropoietin (Epo), GM-CSF, G-CSF, SCF (stem cell factor), multi-CSF (also known as interleukin-3) M-CSF, E-CSF The product according to claim 9, which is (or interleukin-5), IGF-1 (insulin-like growth factor), PDGF (platelet-derived growth factor), or TGF-β2 (transforming growth factor-β2). 11. The product of claim 5, wherein the cytokine or growth factor is a human cytokine or human growth factor and the molecule is functionally equivalent to them. 12. The product according to any one of claims 5 to 10, wherein the proliferative active ingredient is a recombinant human cytokine or human growth factor, optionally modified by one or more amino acid changes. 13. The product according to claim 12, wherein the recombinant human cytokine is recombinant IL-2. 14. The product according to claim 13, wherein the recombinant IL-2 is desala ₁ -IL-2ser ₁₂₅ . 15. The product of any one of claims 1 to 14, wherein the biologically active agent is an antiproliferative agent, a compound that interferes with nucleotide synthesis, a gene sequence, or an antisense nucleotide sequence. 16. The biologically active agent is cyclosporin, FKK506
, Thalidomide, dihydrofolate reductase inhibitor, antiblast agent, platinum coordination complex, vinca alkaloid, purine analog, pyrimidine analog, corticosteroid, viral reverse transcriptase inhibitor, or antisense nucleotide sequence. Item 15. The product according to Item 15. 17. The product according to any one of claims 1 to 14, wherein the biologically active agent is an immunosuppressant, an enzyme inhibitor, an anticancer agent or a radioisotope. 18. The biologically active agent may be cyclosporine, vinca alkaloid, FKK506, thalidomide, methotrexate, azathioprine, cyclophosphamide, actinomycin D, daunonbicin, doxonbicin,
Bleomycin, rhenium radioisotope, yttrium radioisotope, 3 '
15. The product according to any one of claims 1 to 14, which is -azide-3'deoxythymidine, an antisense nucleotide sequence that binds to a viral nucleotide sequence, or an anti-oncogene nucleotide sequence. 19. The ratio of biologically active agent to proliferating active ingredient is 1
The product according to any one of claims 1 to 18, wherein the ratio is greater than 1: 1. 20. A product comprising a biologically active agent bound to a cytokine or growth factor, or a molecule functionally equivalent thereto, wherein the biologically active agent is an anti-inflammatory agent. Selected from the group comprising a proliferating agent, a compound that interferes with nucleotide synthesis, a radioisotope, a gene sequence, an antisense nucleotide sequence, wherein the cytokine or growth factor has a target cell capable of presenting a high affinity receptor for them. Product. 21. The product according to claim 20, wherein the cytokine or growth factor is as defined in any one of claims 6 to 8, 10 to 14. 22. The product according to claim 20, wherein the biologically active agent is an immunosuppressant, an enzyme inhibitor or an anticancer agent. 23. The biologically active agent is cyclosporin, FKK506
, Thalidomide, dihydrofolate reductase inhibitor, antiblast agent, platinum coordination complex, vinca alkaloid, purine analog, pyrimidine analog, corticosteroid, viral reverse transcriptase inhibitor, or antisense nucleotide sequence. Item 22. The product according to item 20 or 21. 24. The biologically active agent is cyclosporine, vinca alkaloid, FKK506, thalidomide, methotrexate, azathioprine, cyclophosphamide, actinomycin D, daunonbicin, doxonbicin,
Bleomycin, rhenium radioisotope, yttrium radioisotope, 3 '
22. The product according to claim 20 or 21, which is -azide-3'deoxythymidine, an antisense nucleotide sequence that binds to a viral nucleotide sequence, or an anti-oncogene nucleotide sequence. 25. The ratio of biologically active agent to proliferating active ingredient is 1
The product according to any of claims 20 to 24, wherein the ratio is greater than: 1. 26. A first domain containing an IL-2 sequence that functions for recognition and growth promotion by a high-affinity IL-2 receptor, and includes an antiproliferative agent, a compound that interferes with nucleotide synthesis, a radioisotope, and a gene. A product which is a molecule comprising a first domain associated with a second domain comprising a biologically active agent selected from the group comprising the sequence and an antisense nucleotide sequence. 27. The ratio of the second domain to the first domain is 1: 1
A product according to any one of the preceding claims, which is larger. 28. A product according to any one of the preceding claims for use as a medicament. 29. The production according to any one of claims 1 to 28 for producing a medicament for treating or preventing a disease or disorder involving cells having a high affinity receptor for a proliferative active ingredient. Use of things. 30. The proliferative active ingredient is IL-2 or a molecule functionally equivalent to IL-2, wherein the disease or disorder is an autoimmune disease, transplant rejection, transplant versus host disease, retroviral disease. 30. The use according to claim 29, which is a lymphoproliferative disorder. 31. A pharmaceutical formulation comprising a product according to any one of claims 1 to 28 formulated for use in medicine. 32. A pharmaceutical composition comprising the product according to any one of claims 1 to 28 and a pharmacologically acceptable diluent, excipient or carrier. 33. Uptake of a biologically active agent into cells having a high affinity receptor for the proliferative active ingredient, cytokine or growth factor of the product of any one of claims 1 to 28; Use of the product for the manufacture of a medicament, optionally for stimulating lymphocyte proliferation. 34. A method for treating or preventing a disease or disorder involving cells having a high-affinity receptor for a biologically active component, comprising a biologically active substance having a high affinity for the receptor. 29. A method comprising administering to a patient an effective amount of the product of any one of claims 1-28, comprising an active agent. 35. A product comprising a biologically active agent combined with a growth active component. 36. A biologically active agent bound to a component that is a peptide hormone having a high affinity receptor or to a component that is a molecule that is functionally equivalent to the peptide hormone with respect to the high affinity receptor. Product comprising.