MXPA00008633A - Utilization of cd137 in order to promote the proliferation of peripheral monocytes - Google Patents

Abstract

The invention relates to the utilization of monocyte growth factor CD137 or of a functional analogue thereof in order to produce a medicament for promoting the proliferation of peripheral monocytes of a mammal. The invention especially relates to the utilization of said growth factor for treating different pathological conditions.

Description

USE OF CD137 TO PROMOTE THE PROLIFERATION OF PERIPHERAL MONOCITS The present invention relates to the use of CD137 monocyte growth factor to promote the proliferation of peripheral monocytes and particularly to the use of CD137 for the treatment of various diseases that can be treated in a novel way through the proliferation promotion action of CD137. Peripheral monocytes of blood and macrophages that originate from them from cavities • Body and tissues are constituents of the body's mononuclear phagocytic system. Particularly, monocytes and macrophages are effector cells of the non-specific immune defense system of the body. The monocytes develop in several intermediate stages from the hematopoietic stem cells in the bone marrow. They circulate in a period of approximately 20 to 30 hours in the blood. From there, they migrate in the various organs and tissue systems and develop there in specific macrophages for sites. Here, the surrounding tissue has a formative influence on them and they develop additional functions. A differentiation is therefore made according to the type of tissue, for example, macrophages of the lung (alveolar macrophages), of the cavity Abdominal (peritoneal macrophages), spleen (splenic macrophages), liver (Kupffer cells), joints, bones (osteoclasts), connective tissue, brain and kidney. Monocytes and macrophages have a central position in the context of inflammatory reactions that occur in the body. In a non-inflamed tissue, the object of. Macrophages is the elimination of old cells. In addition, they produce a large number of soluble factors that are important for communication within the immune system. If the cells are involved, however, in an inflammatory process, they are in an activated state with highly modified phenotypic and functional properties. In this case, they exert important effector functions influencing the course of the disease. They include phagocytosis as well as intracellular destruction of microorganisms, immune complexes and damaged cells, but also antibody-dependent and antibody-independent cytotoxicity reactions against tumor cells, virus-infected cells and parasites. In addition, monocytes and macrophages are of central importance in the induction and regulation of the immune response. That is, they are also very active secretory cells that affect the immune response through the increased release of cytokines. CD137 is a member of the families of tumor necrosis factor receptors and is also known (Kwon, BS, et al., Proc Nati Acad Sci USA 86: 1963, 1989, Schwarz H. et al., Gene 134: 295 , 1993; Alderson, MR, et al., Eur J Im unol 24: 2219, 1994) under the names of ILA or 4-1BB (mouse homologs). CD137 is expressed by activated lymphocytes and monocytes, the expression of primary cells depends on activation (Schwarz H. et al., Blood 85: 1043, 1995). The expression of CD137 is rapidly inducible, for example, by activation of T lymphocytes with phytohemagglutin (PHA) or phorbol-12-myristate-13-acetate (PMA). In monocytes, CD137 is inducible by activation with lipopolysaccharide (LPS), IL-lβ and PMA. In B lymphocytes, the expression of CD137 is induced by antibodies against cell surface immunoglobulin or TMA, and by transformation with EBV (Epstein-Barr virus). In non-lymphoid cells (such as, for example, chondrocytes), CD137 is strongly inducible by the proinflammatory cytosine IL-lß. Soluble forms of CD137 are produced by differential splicing and can be detected in high concentrations in sera from patients with rheumatoid arthritis (Michel, J., et al., Eur J Immunol 28: 290, 1998). The gene for human CD137 is found on chromosome lp36 in a group of related genes (Schwarz, H., et al., Biochem Biophys Res Com 235: 699, 1997). A recombinant CD137 protein is also known which, in immobilized form, causes an activation of monocytes. The results of the activation is an increased expression of proinflammatory cytokines, an inhibition of the anti-inflammatory cytosine IL-fl) and the induction of activation markers, such as ICAM (Langstein J. et al., J. Immunol 160-2488, 1998). An action that prolongs life or that promotes the proliferation of monocytes is not described there. As already mentioned above, monocytes have a function key in the context of the immune response and are of • essential importance for the production of benign immune reactions against tumors and pathogens. Attracted by signals, as for example from the cytosines, migrate from the blood circulation to the site of inflammation. A The accumulation of monocytes and macrophages is a characteristic of chronic inflammation. This accumulation is further promoted by cytokines, which are released at the site of inflammation such as for example the stimulation factor • from macrophage colonies (M-CSF), the stimulation factor of granulocyte-macrophage colonies (GM-CSF), and interleukin-3 (IL-3), which have a favorable effect on the survival of monocytes and macrophages (Young, DA, J Immunol 145: 607, 1990; Z., et al., Am J Respir Cell Mol Biol 6: 212, 1992; Bratton, DL, et al., J Clin Invest 95: 211, 1995).

To date, peripheral monocytes and macrophages were considered to be incapable of proliferation, ie of replication (see, Xing, Z. et al., Supra, van Furth, R., et al., Blood 54-485 , 1979). From the above details, it is clear that the treatment of numerous disorders such as tumors, bacterial, fungal or viral infections, could be significantly improved if an increased phagocytosis and intracellular destruction of microorganisms, immune complexes and damaged cells, and also a Antibody-dependent or antibody-independent cytotoxicity reaction against tumor cells, microorganisms and infected cells could be produced by the replication of monocytes / macrophages. It is also known that various therapeutic forms of treatment, such as for example chemotherapy or radiation therapy of cancer patients, and the administration of immunosuppressants drastically reduce the number of monocytes and macrophages. After the therapeutic procedure of this type is finished, in general terms it is desirable to increase the number of monocytes / macrophages again to values within the normal range and therefore stabilize the patient's immune system again. It is therefore the object of the present invention to provide a pathway that makes possible the specific increase in the number of peripheral monocytes and consequently the number of macrophages, in order to make possible an improved treatment of disease states associated with a number • Inadequate monocytes / active macrophages. 5 Surprisingly it was possible to achieve this object starting from the finding that the cell surface CD137 protein, known per se, and functional analogues thereof, unlike previous assumptions, induce the proliferation of peripheral monocytes independently of hematopoietic stem cells. This surprisingly opens up a large number of new possibilities for therapeutic applications for CD137. Below we present specific embodiments of the present invention in greater detail with reference to the accompanying drawings in which: Figure 1 (A) shows the cDNA sequence and amino acid sequence of human CD137 derived therefrom. A signal peptide (position +1 to +17) and a transmembrane domain (position +187 to 213) are underlined in each case. Sites glycosylation potentials are marked by asterisks; Potential phosphorylation sites (position +242 for protein kinase C, positions +234 and +235 for casein kinase II are also indicated), the polyadenylation signal is shown in bold; (B) an alignment of the sequences of amino acids of human and murine CD137; the identical amino acids are shown by vertical lines; amino acids with a high, low or similarity are marked by two points, point or space. • Figure 2 shows the induction of apoptosis of monocytes 5 by CD137. Figure 3 (A) shows the induction of peripheral monocyte proliferation by immobilized CD137-Fc protein with the aid of 3 H-thymidine incorporation; (B) the formation of monocyte colony induced by CD137. 10 Figure 4 shows the effects of M-CSF and GM-CSF on the • monocyte proliferation induced by CD137; (A) culture of peripheral monocytes on immobilized Fc or CD137-Fc protein in the absence or presence of neutralizing antibodies against M-CSF, GM-CSF and / or IL-3; (B) a The proliferation of monocytes is not induced by M-CSF and GM-CSF; in each case the absorption of 3H-thymidine is shown. Figure 5 shows the induction of (A) growth and (B) proliferation of monocytes by conditioned supernatant of cell cultures that have been cultured with protein CD137-FC. Figure 6 shows a comparison for the induction of proliferation of monocytes by soluble CD137-Fc (sCD137-Fc) and immobilized CD137-Fc; as control, immobilized Fc, soluble Fc (sFc) and untreated support are indicated. Figure 7 shows attempts to induce proliferation of monocytes with Fc, CD137-Fc, TNFR-Fc and anti-CD68. Figure 8 shows attempts to induce proliferation of monocytes with Fc (clear bars) or CD137-Fc (bars • black) in combination with LPS or M-CSF. Figure 9 shows a photographic representation of peripheral monocytes (A) grown in immobilized Fc or CD137-Fc, amplification 400 times; (B) after a 10-day culture in immobilized CD137-FC; Amplification 500 times. Figure 10 shows in the left half of the graph the expression of M-CSF per monocyte after 1 to 8 days of culture in immobilized Fc (white bars) or immobilized CD137-Fc (black bars); and in the right half of the graph the expression of M-CSF in the presence of soluble Fc or soluble CD137-Fc after 8 days of culture. Figure 11 shows the number of live monocytes after neutralization of M-CSF; the number of live monocytes treated with Fc (circle) or CD137-FC (square) is indicated either with 100 ng / ml of M-CSF (triangle) without (clear symbols) or after addition of 2 μg / ml of anti-M-CSF antibody neutralizing (black symbols). Figure 12 shows monocytes after 8 days of culture with a 300-fold amplification in the presence of Fc or soluble or immobilized CD137-Fc protein (in each case 1 μg / ml). The isolation, sequencing and characterization of CD137 Human were first described by Schwarz, H., et al., In Gene (1993), 134, 295, to which reference is made here. The invention relates, as mentioned, to the use of CD137 and "functional analogues" thereof. Functional analogs within the framework of the present invention are particularly variants, derivatives, soluble forms and multimeric forms of CD137 which despite differentiating from the native form of CD137 have the desired biological activity according to the present invention and can therefore be used for the purposes mentioned. Particularly, the functional analogs according to the invention must also have the ability to bind with the target cells, that is, with the peripheral monocytes, in order to be able to induce the proliferation of monocytes in this way. Variants of CD137 include, for example, proteins that can be obtained through one or more amino acid substitutions, removals, additions, insertions and / or inversions, starting from the sequence illustrated in Figure IA. Variants of CD137 according to the invention should have from about 60 to 100%, for example from about 80 to 100% corresponding to the amino acid sequence as shown in Figure IA. Modifications of the native amino acid sequence can be produced in a manner known per se as for example by mutation of the corresponding nucleotides in the nucleotide sequence. Thus, the following can be substituted, for example, among them: amino acids by a similar aliphatic radical, such as for example isoleucine, valine, leucine and alanine; radicals by a similar polar side group, such as for example lysine and arginine; such as glutamine and asparagine, or glutamic acid and aspartic acid. Functional analogues also include non-glycosylated or glycosylated forms other than CD137. The glycosylation pattern may be influenced, for example, by the specific choice of the expression system in the recombinant preparation of CD137. In addition, there is the possibility of specifically modifying the amino acid sequence in the region of potential N-glycosylation sites such that the glycosylation is no longer carried out. Functional analogs further include natural CD137 variants, which can be obtained, for example, by alternative splicing of mRNA either by proteolytic cleavage of CD137 and at the same time can have amino acid sequences truncated at the N-terminus or at the C-terminus. In addition, variants can be obtained by specific removal of these internal or terminal acids or amino acid subsequences that are not of importance for the desired biological function. For example, cysteine radicals can also be specifically removed or replaced in order to avoid the formation of incorrect intermolecular disulfide bridges. Derivatives of CD137 may contain one or more chemical radicals linked through functional side groups of amino acid radicals by chemical or enzymatic modification. Derivatives of CD137 can be obtained by derivatizing amino acid side chain functional groups either at the N-terminus or C-terminus of the protein. For example, glycosyl groups, acyl groups, lipid radicals, phosphate groups, polymer radicals such as, for example, polyethylene glycol side chains, can be introduced in a manner known per se. A particular form of derivation is the N or C end link with another amino acid sequence. Fusion proteins of this type can be prepared both chemically and recombinantly. Analogs according to the present invention also include soluble forms of CD137. These include the extracellular domain of the protein in complete or partial form, while the transmembrane domain is partially or totally removed. However, these forms do not have the cytoplasmic C-terminal sequence portion. In accordance with the invention, the soluble forms of CD137 are especially useful for in vivo applications, since as a result, for example, the intravenous administration of a pharmaceutical preparation is simplified • in a remarkable way. A preferred soluble form of CD137 is a polypeptide with amino acid residues +18 to +186 as shown in Figure IA. Other analogues in the form of soluble or insoluble functional fragments, i.e. partial sequences or combined partial sequences of the native form of CD137, are also included in accordance with the invention. # The soluble forms of CD137 according to the present invention can be prepared in a manner known per se. For example, a recombinant preparation starting from the truncated DNA fragment appropriately is possible.

In addition, there is the possibility of preparing truncated forms of CD137 by specific digestion with protease. Functional analogs of the polypeptide explicitly described in Fig. IA that can be employed in accordance with the present invention also include equivalent polypeptides are functional, such as those that can be isolated from other mammals or other cellular systems of the same mammal. A functional equivalent in this regard is, for example, the factor isolated from mice and described in the US patent specification 5,674,704, which has the name 4-1BB, and the functional analogs in turn derived therefrom as for example fragments. CD137 and its functional analogs described above can be employed in accordance with the present invention both in • monomeric form as in multimeric form. 5 For use in monomeric form, immobilization of the protein is especially convenient. The immobilization can take place, in this case, in a vehicle matrix in a manner known per se. This vehicle matrix can be, for example, the surface of a culture vessel where a cell system containing monocytes can be cultured.

• An additional suitable vehicle matrix can be, for example, polymer particles that can be suspended in a culture system containing monocytes. The optimal number of molecules immobilized per unit area can be easily determined by the person skilled in the art through some preliminary experiments. It can be, for example, from about 1011 to 1017 molecules of CD137 or functional analogs thereof per square centimeter, particularly about 1013 a 1014, as for example about 6 x 1013. The immobilization can be carried out in any usual way known to the person skilled in the art, to the extent that the desired biological activity of the immobilized CD137 molecule is not adversely affected. I do not know is negatively affected only insignificantly as a result. The immobilization can be carried out, for example, by means of the adsorption of the molecule in a vehicle or by covalent bonding with the vehicle, for example, by using difunctional linker molecules linked, for example, by groups. functional amino acid residues. In this case, for example, sources of amide, diazo, isothiocyanate or disulfide can be formed between the linker and CD137 (see, for example, Vitetta et al., 1987, Science 238, 1098; Pastan, et al., 1986, Cell, 47, 641; or et al., 1987, Cancer Res. 47, 5924). An additional possibility of immobilization consists in the expression of CD137 on the cell surface of cells transformed with DNA encoding CD137, for example, CHO cells. There is also the possibility of specifically modifying the amino acid sequence of CD137 in order to facilitate immobilization. Modifications of this type are known, for example, as "markers" and function as anchors, as For example, the modification known as hexa-histidine anchor, or epitopes that can be recognized as antigens by antibodies (described, for example, in Harlow, E. and Lane, D., 1998, Antibodies: A Laboratory Manual, Cold Spring Harbor (NY) Press). These anchors can serve for the fixation of CD137 on a solid support, such as for example a polymer matrix which, for example, may be present on a chromatography column, a microtiter plate or a culture vessel. A particularly desirable embodiment of a functional CD137 analogue suitable for immobilization is a fusion protein from the extracellular domain of CD137 and the Fc part of an IgG molecule. Fusion molecules of this type can be immobilized in a particularly advantageous manner in supports to which either a protein A or an anti-Fc antibody binds. The preparation of CD137-FC is described in Schwarz, H. et al., Blood, 87, 7 (1996), 2839-2845, which is expressly referred to herein. Multimeric CD137 aggregates that can be employed according to the present invention preferably comprise from 2 to 5 molecules of CD137 or functional analogs thereof. The aggregation of the individual peptide molecules must be carried out in such a way that their binding to the monocytes is not prevented. Preferably, the CD137 multimeric aggregates are prepared from the fusion molecules described above that include the extracellular domain of CD137 and the Fc part of an IgG molecule. The Dimerization can be carried out, for example, by cross-linking with an anti-Fc antibody. A dimeric aggregate of CD137 can be obtained in this way. Dimerization is further achieved by the formation of disulfide bridges between the Fc part of two fusion protein molecules. The dimerization is also • possible by the use of bifunctional chemical linkers, for example, those having terminal sulfhydryl groups, such as dithiobis (succinimidyl) propionate, N-succinimidyl 3- (2-pyridyldithio) propionate or reactive carbodiimides such as, for example, l-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride. 10 Higher multimers such as, for example, molecules • having 5 units of CD137, can be obtained from the Fc part of pentameric IgM molecules and the extra cellular domain of CD137. A first subject of the invention relates to the use of the cell surface protein CD137 designated monocyte growth factor, produced by activated T lymphocytes, or a functional analogue thereof for the production of a drug for the promotion of peripheral monocyte proliferation, if appropriate also of progenitor cells and / or precursors (continuation) thereof, of a mammal. A "promotion" of monocyte proliferation within the framework of the present invention includes both the induction of the proliferation of non-proliferating monocytes and Additionally, the aid to monocytes and pluriferantes.

Peripheral monocytes are a constituent of the peripheral blood system and in adults they are found in a concentration of 80 to 540 and in children at a concentration of • 80 to 720 cells per μl of blood under physiological conditions. As is known, all types of diseases are related to a decrease in the number of monocytes and / or cells derived therefrom, particularly macrophages of different tissue specificity (see above). Taking in tells the known physiological functions of monocytes • and cells derived from them, it can be concluded that an increase in the number of monocytes, for example, from a value in the lower physiological range to a value in the higher physiological range would probably be of therapeutic benefit as a result of increased proliferation. Furthermore, it is conceivable that, as a result of the artificial induction of tf monocytosis (increase in the number of monocytes in the blood to values greater than 540 cells per μl of blood), the monocyte body defense is aided during defense against infections. A further subject of the present invention therefore relates to the use of CD137 or a functional analogue thereof for the production of a drug for the treatment of A condition associated with a disorder of a cellular system that includes monocytes and / or cells derived from them and / or progenitors and / or precursors thereof, such as macrophages (see the schematic representation of the system • hematopoietic in Concepts of Gene Therapy, (1997) editorial 5 Walter de Gruyter Berlin, Strauss, M., and Barranger, J.A. (Ed.), Page 236, Table 12.1 to which reference is expressly made here); or whose formation and / or course can be treated by promoting the proliferation of cells in that cellular system. 10 A disorder, in the sense mentioned above, can, in this • case, include an inherited or acquired, permanent or temporary, partial or complete affectation of one or more physiological functions of the body cells in question. Said cellular system is, for example, the cell system myeloids whose cells are derived from progenitor cells from the bone marrow. Typical constituents of this system are granulocytes and monocytes. In particular, the use in accordance with the present invention is indicated when the aforementioned disorder includes a functional disorder or a decrease in the number of monocytes low in a concentration of 80 cells per μl of blood, and / or a decrease in the derived cells. A first preferred field of application for CD137 are conditions in accordance with the invention that are select between damage to the hematopoietic system related to radiation therapy or chemotherapy. Chemotherapy and radiation therapy are frequently carried out for the treatment of all types of oncosis or, in the context of a myelosuppressive or myeloablative therapy, for the preparation of bone marrow or organ transplants. Leukopenia and a decrease in the number of monocytes associated with it is frequently the result. Morbidity and mortality of patients due to an increased susceptibility to infections rises in great measure. Leukopenia could be more effectively • relieved or eliminated by the administration of monocytes treated with CD137 or CD137 according to the invention. An additional field of application refers to the use of CD137 or well functional analogues for the treatment of wound healing disorders as seen, for example, in patients undergoing dialysis either in the case of diabetics or patients with chronic venous insufficiency. In this case, these disorders of wound healing resulting from the granulation tissue insufficiently present or functioning, consisting mainly of macrophages (ie, differentiated monocytes). A further object of the invention relates to the use of CD137 or a functional analog thereof for the production of a drug for the treatment of conditions associated with an inadequate immune response. Examples of conditions of this type that we can relate are the following: a) Oncosis favored by an inadequate cytotoxic activity • absent from the endogenous defense system, 5 b) bacterial, viral or fungal infections favored by an inadequate or absent phagocytosis of the pathogen or body cells infected with it, in addition, the treatment of: c) damage to the immune system or conditions of the immune system acquired or not acquired, inherited or not inherited; and d) damage induced by treatment with immunosuppressants, as may occur, for example, in the case of treatment of patients with chronic polyarthritis or autoimmune disorders of patients undergoing transplants. As will be explained more precisely in a later section, the drug according to the present invention can be used in the context of an in vivo or ex vivo treatment. In accordance with the additional embodiment of the invention, CD137 or a functional analogue thereof can also be used in combination with at least one additional factor that is selected from interleukins, lymphokines, monocins, interferons, colony stimulation factors and growth factors. Non-limiting examples that can mentioned are: IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IFN- α, -β, - ?, TNF-α, EGF, TGF, PDGF, ILGF, MGF, EPO, G-CSF and M-CSF, leukocyte stimulation factors, such as G-CSF, GM-CSF referring to • especially M-CSF. CD-137 and the additional factor can be administered here simultaneously or sequentially in any desired sequence. The combined use with inhibition factors of apoptosis can be conceived. Possibly, M-CSF has an inhibiting effect on this type of monocyte apoptosis. For use in the context of the present invention, CD137 • human is fundamentally adequate in its native form, ie, as a protein having an amino acid sequence of residue +18 to the residue +255 in the sequence according to Figure IA or a functional analog of this sequence. Preferably, however, the extracellular domain of human CD137 corresponding to amino acids +18 to +186 is used as shown in Figure IA or a functional analogue thereof. In a preferred embodiment of the invention, CD137 or its functional analog is employed in immobilized form or as a multimeric aggregate. A preferred multimeric aggregate of CD137 comprises from 2 to 5 protein molecules of CD137 or functional analogues. The invention further relates to an in vitro or ex vivo process for promoting the proliferation of peripheral monocytes, peripheral monocytes from the blood of a mammal by contacting free or immobilized CD137. • particularly immobilized, in nutrient medium and incubated until the number of monocytes has increased, preferably up to its maximum value, and / or until an increase in monocytes can be observed. The isolation of monocytes from the blood of a mammal can be carried out in accordance with methods usual standards. The isolated cell fraction can • be either a pure monocyte fraction or may contain cells that do not adversely affect the treatment of the monocytes and the subsequent treatment of the mammal with the stimulating monocytes. So, for example, lymphocytes may be present during the in vitro incubation in addition to the monocytes. Suitable methods for the isolation of monocytes are described, for example, in Meyskens, F.L. et al, Exp. Haematol. 1979, 7 (8), 401-410; Weiner, R.S., et al., J. Immunol. Methods, 1980, 36 (2), 89-97; and Contreras, T.J., et al., Cell. Im unol. , 1980, 54 (1), 215-229. A non-limiting example of a nutrient medium suitable for in vivo or ex vivo culture is, for example, an RPMI medium supplemented with 5% fetal calf serum. After finishing the culture, it is comfortable to wash the treated fraction of monocytes such as, for example, PBS before their administration again to the mammal to be treated. A further object of the invention relates to the use of CD137 or a functional analogue in a process for the • regenerative treatment of patients treated by chemotherapy or radiation, where a) a fraction of blood containing peripheral monocytes is isolated from the patient's blood before carrying out radiation therapy or chemotherapy, is incubated ex vivo with free CD137 or immobilized particularly immobilized, until the increase in the number of monocytes, or until • reach its optimum and / or until an increase in the size of the monocyte and the blood fraction treated in this way and preferably enriched with monocytes is administered to the patient again after the completion of the therapy; or b) an effective amount of a multimeric CD137 aggregate is administered to the patient before, during or after chemotherapy or radiation therapy to promote the proliferation of endogenous peripheral monocytes. A further object of the present invention relates to the use of CD137 or a functional analogue in a process for promoting the endogenous non-specific immune defense where an amount of CD137 or a functional analogue thereof that promotes the proliferation of peripheral monocytes, In particular, an amount of a multimeric aggregate of CD137 that promotes the proliferation of peripheral monocytes is administered to a patient. This process is particularly suitable to be carried out in patients with tumors and ^ KF in patients suffering from a bacterial, viral or fungal infection. A multimeric CD137 aggregate that can be profitably administered in the context of this process comprises from 2 to 5 protein molecules of CD137 or functional analogs thereof. The administration of these analogues or aggregates comprising the extracellular section of human CD137 corresponding to amino acids +18 to +186 as shown in Figure IA or of a functional analogue thereof capable of binding with monocytes is particularly suitable . In the case of in vivo administration, it is advantageous to use human CD137 or an analogue derived therefrom. If CD137 is administered as a fusion protein, the protein fused to CD137 may also be of human origin. Additional measures to improve the residence time of CD137 in the blood by, for example, PEGylation such as has been successfully used, for example, for other cytokines (for example, PEGylation of G-CSF as described in EP-A-0 335 423), can also be conceived . The particular choice of the dosage of CD137 or of a functional analogue thereof and the particular dosage scheme depends on the decision of the physician. The latter according to the route of administration selected, according to the efficacy of the particular drug, according to the nature and • severity of the condition to be treated, according to the condition of the patient and his response to the therapy will select an appropriate dose and a corresponding dosage scheme. However, a suitable dose would generally be within the range of about 1 to 20 μg / kg of body weight / day or within the range of about 0.01 a l nmol / kg body weight / day. • Based on the blood volume, effective concentrations of CD137 or a functional analogue thereof during therapy may be within a range of about 0.01 μg to 10 μg per milliliter of blood or within a range from about 0.1 pmol to 0.5 pmol per milliliter of blood. For in vitro or ex vivo administration, effective concentrations of CD137 or a functional analogue thereof can be within a range of no more than approximately 0.1 μg / ml of medium particularly more than about 1 μg / ml medium, for example up to about 50 μg / ml medium, or within a range of more than about 1 pmol / ml medium, particularly more that approximately 0.01 nmol / ml of Medium, such as for example about 2 nmol / ml of medium.

The in vitro administration of CD137 and the functional analogues thereof is advantageously carried out using a liquid, parenteral pharmaceutical composition, • particularly intravenous. This composition preferably contains an effective amount of CD137 or a functional analogue thereof, preferably in dissolved form, in a pharmaceutically acceptable carrier that is suitable for this purpose. Examples of acceptable pharmaceutical vehicles are, in particular, aqueous solutions, such as, for example, physiological saline, solution • Saline regulated with phosphate, Ringer's solution, lactated Ringer's solution and the like. In addition, the composition may contain additional additives such as, for example, antioxidants, chelating agents or agents antimicrobials. Oral administration and administration by inhalation are also possible. Intravenous administration is especially convenient if a systemic therapy is required. For the treatment of local conditions, such as, for example, infections When locally restricted, specific subcutaneous or intradermal administration can also be helpful. In the case of the treatment of a local wound, for example, it is possible to employ a superficial cutaneous administration. This can be carried out by applying a solution, of a suspension, an ointment or a gel.

The optimal concentration of CD137 or a functional analogue thereof in the pharmaceutical compositions according to the invention is determined among other things, • through the specific activity of the form CD137 5 used. Suitable proportions by weight of CD137 or of a functional analogue thereof, however, should be within the range of from about 0.0001 to 1% by weight, in particular from 0.0005 to 0.01% by weight, based on the total weight of the composition. The molar concentration, for example, can be found within the range of approximately 1 nmol • at 0.1 nmol, particularly from about 15 to 300 nmol per 100 g of the composition used. The invention further relates to the use of a nucleotide sequence coding for CD137 or an analogue of the same for the production of a gene therapy composition for the treatment of one of the conditions defined above. Genetic therapy compositions of that type comprise a cellular carrier, particularly peripheral monocytes, cells derived therefrom or progenitor cells or precursors of monocytes (see the schematic representation of the hematopoietic system in Concepts of Gene Therapy, (1997) Verlag Walter de Gruyter Berlin, Strauss, M., and Barranger, JA (Ed.) , p.236, Figure 12.1; refers to it is precisely here), wherein the coding nucleotide sequence for CD137 or for a functional analogue thereof is incorporated in a form that can be expressed in a suitable nucleic acid construct. The invention also relates to a process of genetic therapy for the treatment of one of the conditions defined above wherein a composition of genetic therapies according to the invention is administered to the patient. For this purpose, the gene transfer to the cells can be carried out in a manner known per se, • as for example, with the help of viral constructs, non-viral vehicles, such as liposomes or other suitable constructs of nucleic acids (Günzburg, W.H. et al., Gentransfer in Sáugerzellen, [gene transfer in mammalian cells], (1997) Spektrum Akademischer Verlag, Heidelberg, Berlin; Baum, C, et al., Gene Transfer and Transgene Expression in Haematopoietic Cells, in Concepts of (Gene Gene Therapy (1997), Verlag Walter de Gruyter Berlin, Strauss, M., and Barranger, J.A. (Ed.) Pp. 233-256). The nucleic acid construct that can be used in accordance with the present invention is combined, for example, with a virus vector such as for example with an adenovirus vector or an adenovirus vector deficient for replication, or is ligated with a vector of viruses associated with adeno.

An additional advantageous combination is the formation of complete nucleic acid constructs with liposomes. During lipofaction, small vesicles are prepared • Unilamellar from cationic liquids by means of 5 ultrasonic treatment of the liposome suspension. The DNA is ionically bound to the surface of the liposomes, to be precise in such a ratio that they remain in the net positive charge and the DNA forms complexes with 100% of the liposomes. In addition to DOTMA lipid mixtures (bromide of 1, 2-dioleyloxypropyl-3-trimethylammonium) and DOPE • (dioleoylphosphatidyl-ethanolamine), meanwhile numerous novel lipid formulations have been synthesized and tested for their transfection efficiency of several cell lines (Behr, J.P. et al., (1989) Proc. Nati. Acad. Sci USA 86, 6982-6986; Felgner, J.H. et al., (1994) J. Biol. Chem. 269, 2550-2561; Gao, X. & Huang, L. (1991) Biochem. Biophys. Res. Commun. 179, 280-285; Zhou, X. & Huang, L. (1994) Biochem. Biophys. Acta 1189, 195-203). Examples of the novel lipid formulations are DOTAP methylsulfonate N- [1- (2, 3-dioleyloxy) propyl] -N, N, N-trimethylammonium or DOGS (TRANSFECTAM; dioctadecylamidoglycylspermine). In addition to the nucleotide sequence coding for CD137 or a functional analogue thereof, nucleic acid constructs that can be employed in accordance with The invention includes functional regulatory binding, one or more regulatory sequences such as promoters, amplification signals, enhancers, polyadenylation sequences, origins of replication, reporter genes, marker genes. • selectable and similar. Depending on the desired application, this link can lead to an increase or decrease in gene expression. In addition to the newly introduced regulatory sequences, the natural regulatory sequence may still be present before the current structural genes. Through of the genetic modification this natural regulation can optionally be disconnected and the expression of the genes can be increased. The gene construct, however, can also be of simpler construction, ie without insertion of additional regulation signals before the genes structural and the natural promoter with its natural regulation is not removed. Instead, the natural regulatory sequences are mutated in such a way that regulation is no longer carried out and gene expression is increased. Additional helpful regulatory elements can also insert at the 3 'end of the nucleic acid sequences. The nucleic acid sequences may be present in one or more copies in the gene construct. In principle, all natural promoters with their regulatory sequences can be used. In addition, they can be to use synthetic promoters in a profitable manner. Regulatory sequences should preferably make possible the specific expression of nucleic acid sequences. A further variant of treatment forms according to the invention refers to the use of antibodies specific for CD137 or other CD137 antagonists that inhibit or diminish the action of promotion of proliferation of CD137, so that CD137 therapy could optionally be further optimized . The invention thus also relates to antibodies or fragments thereof which can be used for this purpose, which are accessible in a manner known per se, and other correspondingly suitable CD137 antagonists. Conditions accompanied by increased monocyte formation, such as for example certain forms of blood cancer, could optionally be treated with CD137 antagonists of this type. The present invention is explained below in more detail with the help of the following non-limiting working examples. Working Examples Reagents M-CSF, GM-CSF and IL3 were obtained from R &D (Wiesbaden, Germany). Anti-M-CSF; clone 26730.11, purified InG fraction of protein A from ascites fluid from mouse hybridomas. Anti-GM-CSF: clone 3209.1, monoclonal Ind mouse antibody; Anti-IL3: InG fraction purified from protein A from the mouse hybridoma ascites fluid recombinant CD137-FC protein, consisting of the extracellular domain of human CD137 and the constant domain of human immunoglobulin Gi (Fc) was obtained in Alexis (Grunberg, Germany). Human InG1 Fc protein was obtained from Accurate Chemical And Scientific Corporation (Westbury, NY, USA). Reference Example 1: Immobilization of CD137-Fc Polystyrene microtiter plates (Microstest III Tissue Culture Plates; Becton Dickinson, Franklin Lakes NJ, USA) were incubated overnight at a temperature of 4 ° C with a solution of 1 μg / ml of CD137-Fc protein in PBS (phosphate-buffered saline). 50 μi of the solution was used per well. The next morning, the protein solution was refolded and the dishes were washed with PBS. The immobilization of Fc was carried out analogously. Reference Example 2: ELISA ELISA kits were obtained from R & amp;; D Systems (Wiesbaden, Germany)). The test was carried out in accordance with the manufacturer's instructions. Cytokine concentrations were determined in triplicate using the test and expressed as the average value +/- standard deviation. Reference Example 3: Determination of apoptosis of monocytes DNA fragmentation was determined with the help of "Cell Death detection ELISA" (Boehringer Mannheim, Germany) in accordance with the manufacturer's instructions. The measurements were carried out in triplicate. Reference example 4: Isolation and culture of human monocytes Mononuclear cells were isolated from human peripheral blood (PBMC) from leukocyte layers of healthy subjects. For • This, leukocyte layers were diluted with two equal volumes of PBS. An equal volume of Histopaque (Sigma, Deisenhofen, Germany) was covered with a layer. This mixture was centrifuged at 1200 g for 20 minutes. They were isolated PBMC, enriched in the Percoll separation surface in the form of a white layer. The erythrocytes were lysed at room temperature for 2 minutes using 2 ml of 200 mM NH4CL, 10 mM NAHC03, 10 mM EDTA, pH 7.4. The cells were washed twice with PBS formed in pellets at 250 g and resuspended in RPMI medium, supplemented with 5% fetal calf serum. Primary monocytes were isolated therefrom by elutriation (Andreesen, R., et al., J Leukoc Biol 47: 490, 1990). Elutriated monocytes have a purity of 95% and the content of T lymphocytes was less than 3% (estimated through the morphology of the antigen genotype, ie higher expression DE14, DC3, CD4 and CD8). The cells were cultured in polystyrene culture dishes (Becton Dickinson, Franklin Lakes, NJ, USA) in RPMI 1640 medium, supplemented with 5% FCS, in the cell concentration indicated in each case. Reference example 5: Determination of cell proliferation For the measurement of the proliferation of individual cells the "in situ proliferation k'it" was used from Boehringer Mannheim, Germany. 3 x 10 5 cells were inoculated per chamber of an 8 chambered carrier (FALCON, Becton Dickinson, Heidelberg, Germany), which had been coated with Fc protein or CD137-Fc protein and cultured for 10 days. HE added 10 μM of bromodeoxyuridine (BrdU) in 60 minutes. The incorporated BrdU was visualized in accordance with the test instructions by anti-BrdU de-mouse staining and sheep anti-mouse FITC. Monocytes were identified by staining with anti-CD14 antibodies marked with phycoeritin (2 μg / ml; Immunotech, Marseille, France). The chromatin was stained for 5 minutes with 5 μg / ml of Hoechst 33342 (Sigma, Deisenhofen, Germany). The proliferation of cell populations was determined in a 96-well microtiter plate. 105 were used monocytes per well with 0.5 μCi 3H-thymidine for 24 hours, were harvested and measured using a TopCount microplate scintillation counter (Packard, Meriden, Ct, USA). Each batch was counted 3 times and the results are indicated as • average values +/- standard deviations. Example 1: CD137-FC induces apoptosis in monocytes. Primary 105 monocytes were cultured in tissue culture dishes previously coated with a fusion protein consisting of the extracellular domain of CD137 and the constant domain of human hemoglobin Gi (Fc). Plates not treated and plates coated with the Fc protein were used as • controls. In wells coated with CD137-Fc protein, the number of live monocytes on days 1, 3, 5 and 7 of the culture was significantly increased (figure 2 bottom). The degree of apoptosis was determined through the amount of DNA fragmented (figure 2, upper half). Surprisingly, a high degree of apoptosis was found in monocyte cultures that had been treated with CD137-Fc protein. Comparable results were obtained in 3 independent experiments. Example 2: CD137-FC induces the proliferation of peripheral monocytes and causes the formation of monocyte colonies. (a) CD137 induced a strong proliferation of monocytes, which overcompensated the loss of cells by induced apoptosis in a similar manner. 105 monocytes were cultured in 96-well plates wells coated with Fc or CD137-Fc. Proliferation was determined daily by a 24-hour pulse with 0.5 μCi of 3H-thymidine. As the rates of 3H-thymidine incorporation showed, CD137-Fc induced a strong • proliferation of monocytes (figure 3A). Proliferation 5 showed a positive correlation with the culture time of the monocytes in the presence of CD137-Fc protein. The induction of proliferation reached a maximum after 7 to 10 days, the incorporation of 3H-thymidine was increased 30 or more times or more compared to the control cells. It was not observed difference between monocytes in untreated wells and in • Wells coated with Fc protein (results not shown). The substantial increase in the absorption of 3H-thymidine corresponds to the finding in the sense that the proliferation induced by CD137-FC was carried out in the culture in widely distributed form. By labeling monocytes treated with CD137 through a treatment of 1 fc hour with bromodeoxyuridine (BrdU) and subsequent detection with a fluorescein-labeled anti-BrdU antibody, specifically found that 9.35 of the cells (55 +/- 6 of 589 +/- 43) replicated DNA while peripheral monocytes cultured on Fc-coated carriers did not show BrdU incorporation (results not illustrated). Through immunocytochemical investigations it was also It was possible to confirm that the proliferating cells were monocytes and not other blood cells. Specifically, proliferation was determined, in accordance with what was described above by incorporation of BrdU and the identity of the monocytes was verified by simultaneous staining of CD14, a cell surface protein specific for monocytes. The nuclei were made visible by staining with the DNA intercalation dye Hoechst 33342. In these investigations it was found that the proliferating cells presented typical characteristics of monocytes: they were positive for CD14, polynuclear, and showed the typical morphology of monocytes / macrophages (results not illustrated). (b) As the micrograph according to FIG. 3B shows, the treatment according to the invention of monocytes with mobilized CD137-Fc leads to the significant formation of colonies, as has already been shown for other growth factors in the hematopoietic. Example 3: M-CSF and GM-CSF are additional factors essential for the proliferation of monocytes induced by CD137. (a) 105 peripheral monocytes were cultured in immobilized Fc or immobilized CD137-Fc. Neutralizing anti-M-CSF antibodies (2 μg / ml), anti-GM-CSF antibodies (2 μg / ml) and anti-IL-3 antibodies (2 μg / ml) were added according to the plan of the experiment. Proliferation was determined on day 10 through incorporation of JH-thymidine.

It was possible to achieve a complete inhibition of monocyte proliferation through the neutralization of anti-M-CSF and anti-GM-CSF antibodies. The neutralization of • anti-GM-CSF antibody alone caused a decrease of about 18% of the proliferation. The anti-IL-3 antibody had no effect on the proliferation induced by CD137. The addition of anti-GM-CSF and anti-IL-3 antibodies caused a synergistic decrease in proliferation to one third of its original value (see figure 4A). • These results illustrate that M-CSF and GM-CSF were important factors for the proliferation induced by CD137 of peripheral monocytes, (b) In an additional experiment, however, it was found that M-CSF and GM-CSF caused only negligible proliferation at concentrations such as those induced by CD137. Peripheral monocytes were cultured in 96-well plates, coated with Fc protein of CD137-Fc (1 μg / ml) or with M-CSF and GM-CSF in the indicated concentrations (ng / ml). Proliferation was determined on day 10 through the incorporation of 3H-thymidine (Figure 4B). In preliminary experiments, it was found that M-CSF was induced by CD137 at concentrations up to 10 ng / ml. It was not possible to measure the induction of GM-CSF (results not illustrated). In vivo, it was able to detect M-CSF and GM-CSF at concentrations of 10 ng / ml or 50 pg / ml (Kawano, Y., Eur J Haematol 54: 147, 1995; Einer, SG, Curr Eye Res 14: 1045, 1995; Saunders, MA, Br J Pharmacol 120: 545, 1997). In the test series illustrated in Figure 4B, M-CSF and GM-CSF were used at slightly higher concentrations compared to the literature values, namely 100 and 1 ng / ml, respectively. In comparison to CD137, however, it was only possible to induce a fraction of the proliferation caused by M-CSF and / or GM-CSF. This may be taken as an indication that additional factors induced through the action of CD137 on monocytes contribute in an autocrine fashion to the proliferation of monocytes induced by CD137. Example 4: Monocyte proliferation induced by CD137-Fc is mediated by one or several autocrine factors The next thing investigated was whether these factors, which were possibly additionally involved in the proliferation of monocytes induced by CD137, were present in soluble or bound form on the cell surface. For this purpose, peripheral monocytes were cultured in immobilized Fc protein or immobilized CD137-Fc for 24 hours. The cells were then removed by centrifugation at 12,000 g for 5 minutes. 0, 10 and 20 μl of conditioned supernatant were transferred from these cultures to 100 μl of fresh cultures of untreated monocytes. The transfer of this conditioned medium to untreated monocytes from the same donor induced cell growth (Figure 5A) and cell proliferation (Figure 5B) in a dose-dependent manner. To illustrate the effect on cell growth, the cells were photographed at a magnification of 300 times after 8 days. For comparison purposes, the lower illustration in Figure 5A shows monocytes cultured for 8 days in immobilized CD137-Fc protein. To determine proliferation (Figure 5B), Fc or CD137-Fc were immobilized on culture substrate in the manner described above. The monocytes were then cultured with conditioned supernatant at the indicated concentration. As described above, proliferation was determined through the incorporation of 3 H-thymidine. For this purpose, a pulse was carried out with 0.5 μCi of 3H-thymidine for 24 hours on day 8. Repeating the experiment three times led to comparable results. Example 5: immobilization of the CD137 protein is necessary for the induction of monocyte proliferation. Peripheral monocytes were cultured in immobilized Fc and immobilized CD137-Fc and, for comparison, in the presence of Fc and CD137-FC in dissolved form. The immobilization of CD137 and Fc was avoided in the comparison lots by blocking the culture vessels not specifically with bovine serum albumin (incubation with 200 μl of BSA, at 0.1%, for 30 minutes, at room temperature). Proliferation was determined on day 10 by the addition of 3H-thymidine. As the results shown • in Figure 6 illustrate it, the induction of monocyte proliferation by CD137 could only be observed without the CD137 protein being immobilized in advance by coating tissue culture dishes with the protein. On the other hand, if CD137 was administered as a soluble protein, the induction of proliferation was markedly diminished too. In three experiments • independent, three comparable results were obtained. Example 6: Investigation of monocyte proliferation in the presence of TNFR-Fc and anti-CD68. Fc protein, CD137-Fc protein, factor receptor tumor necrosis (TNFR) -Fc protein and anti-CD68 were immobilized for further characterization of the induction of monocyte proliferation. The incorporation of 3 H-flfe thymidine was determined in accordance with that described above. It is evident from Figure 7 that only CD137-Fc significantly induces the proliferation of monocytes. TNFR-Fc and anti-CD68 are both proteins that bind on the surface of monocytes in a similar way to CD137. With its use as a control, it must be excluded that only the link of monocytes on the surface of the culture vessel cause the observed effects. Example 7: CD137 and M-CSF additively induce monocyte proliferation. • 105 were introduced per well into a 96-well microtiter plate. Each well had been previously coated with Fc or CD137-Fc (in each case 1 μg / ml). M-CSF (100 μg / ml) and LPS (lipopolysaccharide) (50 μg / ml) in dissolved form were added. Measurement of proliferation after cultivation for 8 days was carried out by means of the incorporation of 3H-10 thymidine after a 24-hour pulse with 0.5 μCi of 3H-thymidine. As shown by the experimental results in Figure 8, the incorporation of 3H-thymidine into monocytes is additively increased by immobilized CD137-Fc or M-15 CSF. On the other hand, LPS does not cause any significant increase in the rate of incorporation, and therefore has no effect on monocyte proliferation induced by CD137. Example 8: CD137-Fc induces morphological changes in the monocytes. Primary peripheral monocytes were cultured in untreated tissue culture dishes or immobilized CD13 7-Fc or Fc protein (1 μg / ml). The cultures were photographed on days 1, 2, 4, 6 and 10 with a magnification of 400 times (figure 9A). The culture plates that had only been treated with Fc protein showed only a slight adherence of the monocytes. However, monocytes retained their round morphology. Over the course of 10 days, the monocytes in this lot gradually died (Figure 9A, left column). A totally different result is obtained, however, if the cells have been cultured in tissue culture plates in which a CD137-Fc fusion protein had been immobilized. CD137-FC induces an adherence of monocytes. These cells gradually took an irregular shape. This effect could already be observed on day 1. With the increase of the culture period, the cells expanded, increased in size and presented a more complex morphology (Figure 9A, right column). On day 10 of the culture in the presence of immobilized CD137-Fc, it was possible to identify three monocyte morphologies that differentiated in vitro: an elongated shape (E), a round shape (R) and a branched form (B) (see figure 9B) ). Example 9: CD137-Fc induces the formation of M-CSF (a) 105 primary monocytes were cultured in immobilized CD137-Fc or Fc (in each case 1 μg / ml). In order to maintain the CD137-Fc protein (1 μg / ml) in solution, immobilization of CD137-Fc protein was prevented in a batch by preincubation of the tissue culture plates with fetal calf serum (1 hour at 4 ° C). C, undiluted). The results appear in figure 10. Culture supernatants were harvested at the indicated times and the concentration of M-CSF was determined through ELISA. Comparable results were obtained in three separate experiments. 5 As shown in figure 10, CD137 induces the expression of M-CSF. During the first three days of culture, the M-CSF contents were low and no difference was observed between the control monocytes and the monocytes treated with CD137. From day 4, the formation of M-10 CSF was detectable and the concentration of M-CSF rose rapidly and continuously. Immobilization of the CD137 protein was a necessary prerequisite for a significant formation of M-CF, since soluble CD137 induced only low concentrations of M-CSF. 15 (b) In a next experiment, it was shown that the expression of M-CSF is not only induced by CD137, but is also essential for the survival of induced monocytes ^ fc by CD137. For this purpose, the monocytes were cultured in the presence of immobilized CD137 protein and neutralizing anti-M-CSF antibodies. It was observed that approximately ten times more monocytes survived on plates coated with CD137-Fc compared to plates coated with Fc protein. The addition of neutralizing anti-M-CSF antibodies decreased the number of monocytes survivors to the base value (see table 1). Upon observing the time course, it was found that most of the cells cultured in plates not coated or coated with Fc had already died on day 6 (see figure). • eleven) . Immobilized Cdl37 protein or M-CSF, however, stabilized the number of cells at a high level even on day 12. After capturing M-CSF with the help of neutralizing antibodies, this effect was no longer observed. It is known that in addition to M-CSF, GM-CSF and IL-3 in humans also positively affect the life time of monocytes.

It was not possible to find any expression of GM-CSF or IL-3 in ELISA after culture for 8 days (in the control batch and in the cells treated with CD137) in the experiments according to the invention (results not illustrated) . Neutralization of anti-GM-CSF antibodies, however, the monocyte survival rate mediated by CD137 decreased by half (see table 1). The anti-Il-3 antibodies had no effect. A combination of neutralizing anti-IL-3 antibodies and anti-GM-CSF antibodies, however, decreased the number of monocytes survivors almost to the base value. This shows that IL-3 also plays an important role for the survival of monocytes. The neutralization of GM-CSF and / or IL-3 in addition to the neutralization of M-CSF does not cause any further decrease in the number of cells.

It was possible to decrease the positive effect of M-CSF completely by administering neutralizing anti-M-CSF antibodies and almost to the base value through a combination of anti-GM-CSF and anti-IL-3 antibodies. • Table 1 5 Antibody stimulus no. of cells value p4 > neutralizing2 'survivors11 Fc b.o 1.0 CD137-FC 75.5 ± 6.8 CD137-FC a-M-CSF 9.3 ± 2.0 0.005 10 CD137-FC a-IL-3 72.3 ± 8.4 n. s. • CD137-FC a-GM-CSF 36.0 ± 2.5 0.006 CD137-FC aM-CSF + -IL-3 7.0 ± 1.8 0.005 CD137-FC aM-CSF + a-GM-CSF 6.0 ± 3.0 0.007 CD137-FC a-GM -CSF + a-IL-3 19.0 ± 3.0 0.005 15 CD137-FC a-GM-CSF + a-GM-CSF 4 1..88 ±± 33..33 0.008 + a-IL-3 M-CSF 68.3 ± 16.2 M-CSF aM-CSF 5.3 ± 2.6 0.006 M-CSF a-GM-CSF + a-IL-3 20.8 ± 5.0 0.018 20 1 primary peripheral monocytes were cultured on immobilized Fc protein or CD137-Fc protein (1 μg / ml) or M-CSF was added in a final concentration of 100 ng / ml. 21 the neutralizing antibodies were used in a concentration of 2 μg / ml; the addition was carried out at the beginning of the experiment. 31 average number of cells and standard deviation. On day 10, the cells were counted in four representative fields of the plate. 4) the p values were determined by the Student test with the help of programmatic SSPS. n.s. = not significant Example 10: Immobilization of CD137 protein increases the number of live monocytes. Peripheral monocytes were cultured in immobilized Fc or CD137-Fc protein (in each case 1 μg / ml) or in the presence of soluble Fc or soluble CD137-Fc protein (FIG. in each case 1 μg / ml). The cultures were photographed on day 8 with an enlargement of 300 times (see figure 12). Immobilization was carried out by pre-treatment of the culture plate with bovine serum albumin (30 minutes at room temperature, 200 μl per well of a 96-well plate). In the presence of soluble proteins, the number of monocytes decreases markedly. In accordance with this finding, M-CSF is induced only if the CD137 protein is present in immobilized form (see example 9). It is therefore likely that CD137 will only act on monocytes when a ligand of CD137 expressed by monocytes (ie, a binding partner for CD137) is cross-linked through the binding of immobilized CD137 molecules.

Soluble CD137-Fc secondarily cross-linked through anti-Fc antibodies also prolongs the survival of monocytes, which is observed after culturing for 8 days (results not illustrated). For this experiment, 5 CD137-Fc (2 μg / ml) had been treated in advance with goat anti-human Fc antibody (2 μg / ml). • fifteen twenty

Claims (21)

1. CLAIMS 1. The use of CD137 monocyte growth factor or a functional analog thereof for the production of a • drug for the promotion of the proliferation of 5 peripheral monocytes of a mammal.
2. 2. The use of CD137 monocyte growth factor or a functional analogue thereof for the production of a medicament for the treatment of a condition associated with a disorder of a cellular system that includes 10 monocytes and / or cells derived therefrom and / or • parents and / or precursors thereof; or whose formation and / or course can be treated by promoting the proliferation of cells of this cellular system.
3. 3. The use according to claim 2, wherein the cell system disorder includes a decrease in peripheral monocytes and / or cells derived therefrom or functional disorder of monocytes. { Peripherals and / or cells derived therefrom.
4. 4. The use according to claim 2, for the treatment of conditions associated with an inadequate immune response.
5. The use according to one of claims 2 to 4, wherein the condition is selected from a) damage to the hematopoietic system related to chemotherapy or radiation therapy; b) wound healing disorders; c) oncosis; d) bacterial, viral or fungal infections; e) inherited or non-inherited damage, acquired or not acquired to the immune system or conditions of the immune system; and f) damage induced by treatment with immunosuppressants.
6. 6. The use according to one of the preceding claims, wherein an in vivo or ex vivo treatment is carried out.
7. The use according to one of the preceding claims, wherein CD137 or a functional analogue thereof is used in combination with at least one additional factor that is selected from interleukin, lymphokines, monocins, interferons, colony stimulation factors and growth factors.
8. 8. The use according to claim 7, wherein the additional factor is a leukocyte stimulation factor.
9. 9. The use according to claim 8, wherein the factor is selected from G-CSF, GM-CSF and M-CSF.
10. The use according to one of the preceding claims, wherein CD137 or its functional analogue is used in immobilized form or as a multimeric aggregate.
11. The use according to claim 10, wherein the multimeric aggregate of CD137 comprises two to three protein molecules of CD137 or functional analogs thereof.
12. 12. The use according to claim 10 or 11, • where the extracellular section of human CD137 is used 5 which corresponds to amino acids +18 to +1876 in accordance with Figure IA or a functional analog thereof capable of binding monocytes.
13. 13. An in vitro process to promote the proliferation of peripheral monocytes, peripheral monocytes of the blood 10 of a mammal contacted with CD137 in a medium • nutrient and incubated until the increase in the number and / or size of monocytes.
14. 14. A process for the regenerative treatment of patients undergoing chemotherapy or radiation therapy, where 15 a) a fraction of blood containing peripheral monocytes is isolated from the blood of the patient before carrying out the chemotherapy or radiation therapy, said fraction is incubated ex vivo with CD137 until the increase in the number and / or size of the the 20 monocytes, and the blood fraction treated in this way is administered to the patient again after the therapy is finished; or b) an effective amount of a multimeric CD137 aggregate is administered to the patient before, during or after 25 conclude chemotherapy or radiation therapy to promote the proliferation of endogenous peripheral monocytes.
15. A process for the promotion of endogenous non-specific immune defense wherein a quantity of CD137 or a functional analogue thereof that promotes the proliferation of peripheral monocytes is administered to a patient.
16. A process according to claim 15, wherein the patient is a patient having a tumor, suffering from a wound healing disorder, from a bacterial, viral or fungal infection, from inherited or non-inherited, acquired or non-acquired damage. to the immune system; or of damage induced by treatment with immunosuppressants.
17. A process according to any of claims 14 to 16, wherein the multimeric aggregate of CD137 comprises two to five protein molecules of CD137 or functional analogs thereof.
18. A process according to one of claims 14 to 17, wherein the extracellular section of human CD137 corresponding to amino acids +18 to +186 according to Figure IA or a functional analogue thereof capable of binding with monocytes is employed. .
19. The use of a nucleotide sequence coding for CD137 or a functional analogue thereof to prepare a gene therapy composition for the treatment of one of the disorders defined in one of claims 2 to 5.
20. 20. A gene therapy composition, which comprises a cellular substrate in which it is incorporated in a way that • the coding nucleotide sequence can be expressed 5 for CD137 or for a functional analogue thereof.
21. 21. A gene therapy process for the treatment of one of the disorders defined in one of claims 2 to 5, wherein a gene therapy composition according to claim 20 is administered at ^ Faith 10 patient. fifteen • twenty 25