MXPA97003626A - Method for manufacturing a medicine to treat second secondary immunodeficiency - Google Patents

Abstract

The present invention relates to a method for producing a natural cytokine mixture, it is described including the steps of immobilizing at least one nitrogen in a tissue culture vessel. An isolated population of lymphocytes free of neutrophils and erythrocytes is suspended in serum-free media and placed in the container. Lymphocytes are cultivated, the media removed and characterized for the production of cytokines

Description

METHOD FOR MANUFACTURING A MEDICINE FOR TREATING SECONDARY IMMUNODEFICIENCY DESCRIPTION OF THE INVENTION The present invention relates to an improved method of producing a natural mixture of cytokines. In recent years, it has become possible to modulate the immune system to improve its response and, where the system components are not functioning, either partially or completely restore the function of the component. For example, bone marrow transplantation is used to replace progenitor cells or provide lost progenitor cells to cure the combined, severe immunodeficiency. In another example, immune cells are removed from cancer patients, treated and returned to the patient, in which there is regression of the tumor (Hwu and Rosenberg, 1994a, Hwu and Rosenberg, 1994b). In addition, components of the humoral immune system such as gamma-globulin and intravenous immunoglobulin (IVIG) are finding wide therapeutic applications (DeSimone et al., 1990, Hall, 1993). Other components of the immune system are also being used as therapeutic agents. (Hadden &Smith, 1992; Hadden, 1993; Talmadge and Hadden, 1994). Immunoregulators are compounds that modify immune function or have a positive or negative effect on the activity of the immune system. The use of immunoregulators in clinical medicine includes the reconstitution of the immune function (or the correction of the one in deficiency) and the suppression of normal or excessive immune function. A major class of immunoregulators is that of cytokines. By means of recombinant technology, many of the cytokines are now available for clinical use. However, the immune system is complex and the interaction of several components is often necessary to effectively modify immune functions. It would be useful to design preparations that provide the various components and interactions to effectively regulate immune function. Cytokines are peptide / protein immunoregulators that are produced by activated immune cells, including T lymphocytes derived from the thymus (T cells), B lymphocytes and monocytes / macrophages. Cytokines include interleukins (IL-1 to IL-15), factors that stimulate colony formation (CSF) for granulocytes and / or macrophages (CSF-G, CSF-M, CSF-GM), tumor necrosis factors ( TNF a and ß) and interferons (IFN a, ß and gamma). Interleukin-2 (IL-2) is a lymphokine initially described as a factor in the growth of T cells (Morgan et al., 1976). Chemically, IL-2 is a glycoprotein of 133 amino acids, with a molecular weight of 15,000 Dalton units. It is produced by lymphocytes of normal peripheral blood, by splenic and tonsillar T lymphocytes and by large granular lymphocytes. IL-2 induces and supports the proliferation of T cells stimulated by antigen or mitogen. In addition to the function of stimulating T lymphocytes, IL-2 is important in such processes as the initiation, expansion and regulation of the immune response, the production of gamma-interferon (IFNgamma), the induction of lymphocyte-activated killer cells ( LAK), the propagation of cytolytic T cells and the increase in the destructive activity of natural killer cells (NK). Recombinant IL-2 (rIL-2) is a non-glycosylated protein that is produced by the human cDNA sequence. IL-2 engineered can be obtained as described by Taniguchi et al., (1983); and Devos, (1983) and United States Patents 4,604,327 and 4,569,790. A rIL-2 mutein, in which the cysteine at position 125 of the native or wild type molecule has been substituted with a neutral amino acid, for example alanine or serine, can be obtained as described in U.S. Pat. 4,518,584 de Mar, et al. A method for producing and isolating rIL-2 at a clinical purity can be found in U.S. Patent No. 4,569,790 to Koths et al., As well as the isolation of native IL-2 from cultured T cells. Methods for producing natural IL-2 can also be found in U.S. Patent Nos. 4,390,623; 4,464,355; and 4,448,879. Thurman et al. (1986) describe that different preparations of natural, partially purified IL-2 (nIL-2) and rIL-2 preparations vary significantly in their activity in several biological assays. Several individual cytokines, both natural and recombinant, have been investigated for the treatment of cancer and other diseases. For example, the recombinant interferon ar2 (rIFN a2) is approved by the United States Food and Drug Administration (FDA) for the treatment of Hairy cell leukemia, Kaposi's sarcoma, condyloma acumenata and chronic hepatitis. The IFNc. natural, as a mixture (AlferonR) of twenty or more made by leukocytes, is authorized for condyloma acumenata. Recombinant IFN-gamma (rIFN-gamma) is licensed for chronic atomic granule disease. RIL-2 is authorized for renal cell cancer. These and other rlL and rIFN are under active evaluation and a variety of diseases that include various forms of cancer. In addition, cancer therapy with rIL-2 has been explored in many clinical and research centers. Rosenberg and colleagues (Rosenberg et al., 1985, Mulé and Rosenberg, 1987, Rosenberg, 1988, Belldegrun and Rosenberg, 1989, Chang and Rosenberg, 1989, Rosenberg, 1994) have reported the use of rIL-2 administered systemically in immunotherapy. of patients with renal cell cancer, and malignant melanoma. Cortesina et al. (1988, 1994) described the effects of loco-regional injections of rIL-2 and natural in patients with head and neck cancer and found that natural IL-2 is more effective in causing tumor regression. Patients given large doses of rIL-2 have been toxic and life threatening (Rosenberg et al., 1994). The development and commercial availability of immunoregulators manipulated by genetic engineering (recombinant) has accelerated the evaluation of these agents in the clinic against cancer. The limited efficacy and significant toxicity associated with high doses of rIL-2, rIFN-gamma, rTNF-a and other onotherapies, suggests the reconsideration of natural combinations of cytokines in therapeutic strategies. In addition, more than one hundred different cytokine activities have been identified, which increases the significant doubt as to whether immunotherapy, based on combining recombinant cytokines, has a reasonable probability of success in the clinical treatment of cancer in the near future.

For example, although IL-2 can stimulate T lymphocyte proliferation as a T cell growth factor, many other factors including other interleukins and thymic peptides are produced in the thymus and are also considered necessary for the development and function of T-cells. of the T lymphocytes (Hadden, 1992). An uncharacterized preparation referred to as well as a natural interleukin (NI) preparation has been shown by the applicant to be effective in promoting the development of T lymphocytes. This mixed preparation is not characterized (also referred to as inflammatory membrane coating interleukin, BC). -IL) stimulates the proliferation of protocytes, immature and mature thymocytes in vi tro more effectively than an equivalent concentration of rIL-2 (Hadden et al., 1989). This preparation of NI increased the development of T lymphocytes in neonatal mice, whereas rIL-2 was inactive (Hadden et al., 1989). In addition, this preparation of NI increased the development and function of T lymphocytes in old mice treated with hydrocortisone, while rIL-2 in the equivalent dose was inactive (Hadden et al., 1992). In addition, a mixture of NI without characterizing in low dose prolonged life in mice carrying malignant melanoma; rIL-2 in the equivalent dose was inactive (Kameda et al., 1992). These findings indicate that mixtures of interleukins have activities not provided by IL-2. Attempts to correct T lymphocyte defects have been treated experimentally in a variety of settings including decreased T lymphocytes (lymphocytopenia) and T lymphocyte dysfunction (anergy) that occurs in aging, cancer, AIDS and other immunodeficiencies. For example, rIL-2 and thymic peptides have been used in infection with the AIDS virus (HIV) with variable results (Hadden, 1991). High doses of rIL-2 by continuous infusion has been shown to increase transiently T lymphocyte counts in blood of patients with HIV infection but with considerable toxicity (Lane and Fauci, 1985). Pegylated rIL-2 in one and three million units produced less toxicity, but only side effects on lymphocyte counts in humans with HIV infection (Merigan, 1993). An NI preparation significantly increased T-lymphocyte counts in patients with lymphocytopenia cancer without toxicity (Hadden et al., 1994). These findings indicate that natural interleukins act in humans at low doses to increase T cells without toxicity and that rIL-2 although active at high doses is too toxic for medical use. These findings also support the extrapolation of murine data to man.

This suggests that the use of cytokine preparations that occur naturally may be more efficient in affecting the immune system with less toxicity. However, the preparations that are currently available are not well characterized and are troublesome to produce as described in the following. In order to reproducibly modulate the immune system, it would be useful to have well characterized serum and mitogen free cytokine preparations that can be produced easily and cheaply and from which it will be possible to establish the preparation at low toxicity reproducible for clinical use. U.S. Patent No. 4,985,241 to Zimmerman et al., Discloses the use of a recombinant lymphokine or cytotoxin in combination with a biological modifier, such as a free radical scavenger or a metabolic inhibitor in the therapeutic and prophylactic treatment of damage Biological challenge caused to mammalian hosts by the generation of free radicals, but does not suggest the production of a defined mixture of cytokines that occur naturally. Therefore, it would be useful to produce a natural cytokine mixture (NCM) of lymphocytes that can be used therapeutically in the treatment of diseases and other conditions, which include a function, development and reduced number of T lymphocytes, that is ~ cellular immune deficiency. To be therapeutically useful, NCMs must be sterile, free of endotoxins, without serum, without mitogen, without virus and without DNA to avoid reactions of the receptors. Molecular sieving techniques can eliminate many of these contaminants. However, the most necessary procedures for production, the higher cost. Additionally, more management steps required, lower performance as well as opportunities to increase pollution. Therefore, the NCM can be produced in such a way as to minimize or eliminate any of these contaminants, production will be more cost effective. The preparation of NI used in the studies described in the above, was without serum and without mitogen, but it had to be concentrated lOx before being used. Natural interleukin-2 made as taught in U.S. Patent Nos. 4,390,623 and 4,464,355 was generated in serum-free media, so it does not require the steps to remove serum proteins. The preparation was described as natural interleukin-2, without mitogen, without serum having important activity for cancer. This material was prepared by exposing the cells only briefly to the mitogen (a pulse technique) originally described by Hadden et al., (1970).

The pulse technique is used to stimulate the lymphocytes in tissue culture and to avoid having the mitogen present in the media, when they are harvested. The cells are initially cultured for two hours in the presence of a mitogen in a serum-free medium. After this incubation, the cells are re-isolated and washed three times in media that do not contain the mitogen and then resuspend at a lower density in medium without fresh tissue culture serum without mitogen. The preparation was not characterized in terms of its components other than IL-2. U.S. Patent No. 4,448,879 to Fabricius et al. Also teaches a cell culture process to produce a non-mitogenic, serum-free IL-2, natural. The method used cells with inflammatory membrane coating in a rotary culture system, or in a system that recirculates the media mechanically. However, the method still requires a stage in which the cells are washed until they are free of mitogen and serum and then recultivated in media without mitogen, without serum. Importantly, the described methods are only ineffective for stimulating cells and producing low IL-2 yields. The large volumes required are expensive and require manipulation with skill, extensive and must be concentrated before being used resulting in loss of activity (approximately 50%).

Martorell, et al (1987) provides a method of inducing itogenesis with the continued presence of mitogen in media containing serum. In its method the performance of IL-2 is extremely low and requires the presence of serum. It would be useful to have a method, which does not require the step of washing the cells to leave them free of serum and mitogen and does not require expensive equipment. In the systems described in the foregoing, mitogens are generally plant lectins such as phytohemagglutinin (PHA) and concanavalin A (ConA) which have affinity with the T cell antigen receptor (TCR) (Chilson and Kelly-Chilson, 1989 ) and has a mitogenic effect for T lymphocytes. Exposure of T lymphocytes to such lectins stimulates the production of natural cytokines. In the absence of serum in culture media, generally only low levels of cytokines, particularly interleukins, are produced. For example, IL-2 is generally only in the range of 0-20 units / ml (units per milliliter) under serum-free culture conditions (U.S. Patent Nos. 4,390,623 and 4,464,355). With serum, the range of IL-2 production is generally 10 or more units per ml.

It should be useful to stimulate the lymphocytes to produce cytokines such as IL-2 at higher levels in the absence of serum, such that the mixture can be used more efficiently as a therapeutic agent and without the added stage and decreased yield of cell washing after a pulse exposure for mitogen or using specialized equipment to concentrate the preparation with the associated loss of activity. The present invention provides a unique method for producing a mixture of natural cytokine by the immunization steps of at least one mitogen in a tissue culture vessel. A population . isolated from neutrophil and erythrocyte-free lymphocytes is suspended in medium without serum and placed in the container. The lymphocytes are cultured, the media without mitogen and without serum are removed and characterized for the performance of the cytokines. BRIEF DESCRIPTION OF THE DRAWINGS Other advantages of the present invention will be readily appreciated as they come to be better understood by reference to the following detailed description, when considered in relation to the accompanying figures, in which: FIGURE 1 is a bar graph showing the content of ILs (expressed as optical density of IL-2 (IL2) measured by ELISA) of NCM in X-vivo-10 (EX10), X-vivo-15 (EX, 5), X-vivo-20 (EX2Q) and minimum essential medium (MEM) and compare continuous exposure (empty bar) with pulse exposure (cross-hatched) to the PHA mitogen; FIGURE 2 is a bar graph showing the effect of cell concentration on the generation of NCM with PHA at 1 μg / ml in different media, 1 x 106 / ml cells (cross-hatched), 2.5 x 106 / ml of cells (diagonal lines) and 5 x 106 / ml of cells (empty bar); FIGURE 3 is a bar graph showing the effect of cell concentration on the generation of NCM with a PHA at 2 μ / ml in different media, 1 x 106 / ml of cells (cross-hatched), 2.5 x lQ ^ / ml of cells (diagonal lines) and 5 x 106 / ml of cells (empty bar); FIGURE 4 is a dose-response graph showing the effect on thymidine incorporation (stimulation with ConA) of in vitro treatment of splenocytes from mice without involvement with NCM (solid line) compared to recombinant IL-2 (dotted line) at equivalent concentrations of IL-2; FIGURE 5 is a dose-response plot showing the effect on thymidine incorporation of in vitro treatment of mouse thymocytes without involvement with NCM (solid line) compared to recombinant IL-2 (dotted line) at equivalent concentrations of IL-2; FIGURE 6 is a bar graph of splenocyte responses in vi tro a -rIL-1 (empty bar), rIL-2 (full bar), NCM (cross-hatched) and concanavalin A (diagonal lines) after treatment in vivo with rIL-1, rIL-2, rIL-1 + rIL-2 or NCM; FIGURE 7 is a bar graph in response of thymocytes in vi tro to rIL-1, rIL-2, NCM and ConA after in vivo treatment as in Figure 6; FIGURE 8 is a bar graph showing the effect of in vivo treatment of control mice (open bar) or NCM (filled bar) on the markers of the splenocytes for CDR ", CD8 + and CD4" / CD8"cells (- -), FIGURE 9 is a bar graph showing the effect of in vivo treatment of mice with control (empty bar) or NCM (full bar) on thymocyte markers for CD4_ / CD8 cells "(- -), CD4 + / CD8 + cells (++) and CD4"and CD4 + (CD4 + CD8) cells, FIGURE 10 is a bar graph of thymocyte responses in vi tro to IL-1, IL-2 and NCM after in vivo treatment with control means (empty bar) and NCM (filled bar), FIGURE 11 is a bar graph of splenocyte responses as in Figure 10, FIGURE 12 is a bar graph of thymocyte responses in vi tro to ConA and PHA after in vivo treatment with control means (empty bar) and NCM (filled bar), and FIGURE 13 is a bar graph of the splenocyte responses as in Figure 12. The present invention provides a method of reproducibly producing a mixture of characterized natural cytokine (NCM), ie a cell culture supernatant containing multiple cytokines.The assembled lymphocytes, generally of an inflammatory membrane coating, free of neutrophils and erythrocytes of multiple negative donors hepatitis viruses, negative for HIV are used to produce the cytokine mixture of the present invention. The use of multiple donors takes advantage of the response of the mixed lymphocytes (MLR). In addition, in a preferred embodiment up to fifteen donors could be used each time to produce the mixture, to ensure that the MLR response is constant for each preparation and any variation. In an alternative modality, autologous lymphocytes would be used to generate the NCM. In these cases, the patient should not be without a virus. In addition, if autologous lymphocytes are used, the patient may be returned as necessary. In an alternative embodiment, the animals could be the source of cells for veterinary uses.

The lymphocytes are cultured in the presence of immobilized mitogens in a tissue culture vessel. In a preferred embodiment, the mitogen is immobilized on the surface-activated cell culture flasks for the selection of cell subclasses (T-25 AIS microCELector ™ plates) as described in the manufacturer's instructions. However, other methods of immobilizing mitogens on the surface of the culture vessel, such as methods of incorporating other "expansion" or coupling techniques into sepharose 4B beads can be used as are well known in the art of cell isolation. The use of immobilizing cells for selection is well known in the art; however, the use for cytokine generation is new. Mitogens are generally selected from lectins and monoclonal antibodies that stimulate lymphocytes to produce cytokines. In a preferred embodiment, phytohemagglutinin (PHA) or OKT3 are used (OrthocloneR, Ortho Pharmaceuticals). Other lectins such as concanavalin A (ConA) or the phytolac mitogen which stimulates B cells can be used. Monoclonal antibodies to T cell receptors such as CD2, CD28, CD45 can be used as mitogens and would be effective. Antibodies against CD28 and CD45 are reported to be hyperproducers of IL-2 (Deans et al., 1989 and June et al., 1989). In addition, antilymphocyte globulin (ALG) has mitogenic activity for T cells. In addition, combinations of mitogens could be used to activate a combination of lymphocyte subpopulations. The PHA is used in the preferred embodiments and is coated at an initial concentration of approximately 25 μg / ml. The lymphocytes were incubated for 24-48 hours in serum-free media with continuous exposure to mitogen, ie without washes. In a preferred embodiment, the media is either X vivo-10 or X vivo-15 media (Whittaker). This is a serum-free medium and approved by the FDA for IL-2 / LAK infusions in patients as stated in the manufacturer's catalog. Serum-free media capable of supporting the proliferation of human lymphocytes such as RPMI-1640 (Sigma) can also be used. The media also contains a 4-aminoquinolone antibiotic. In the preferred embodiment, the antibiotic is ciprofloxacin. The antibiotic is used to maintain sterility and for overproduction of lymphokines. Ciprofloxacin and related antibiotics have been reported to increase IL-2 and other cytokines in the presence of soluble mitogen and serum. (Riesenbeck et al., 1994). It has not been reported that they are effective in the absence of serum. Its use with immobilized mitogens is also new. Ciprofloxacin is used in the preferred embodiment at a concentration of about 20 to about 200 μg / ml and most preferably at a concentration of about 80 μg / ml. The supernatant is removed and is the source of the natural cytokine mixture (NCM) of the present invention. The supernatant is free of mitogen as shown in Example 1 and in animal studies and in initial humans they do not have to be concentrated. Human serum albumin (HSA) can be added to stabilize the NCM in the supernatants. HSA is used in place of serum albumin from a non-human source, because the HSA has been approved by the FDA for human use. A cytokine profile of the supernatant is established using the following assays. The interleukin (IL) content of the supernatants is confirmed by the bioassay for IL-2 and by ELISA for other interleukins, CSF, TNF and IFN. Sterility is tested and endotoxin is measured by the lysis lysate assay. Specifically, the following assays and kits are used in a preferred embodiment: INF-gamma ELISA (ENDOGEN), IL-1, IL-2, IL-3, IL-4, IL-6, IL-7, IL-8, GM-CSF, G-CSF and TNF-a ELISA (R &D Systems). The IL-2 bioassay is by the method of Gillis et al., (1978) and is expressed as units / ml compared to a known standard of IL-2 (Schiapparelli Biosystems, Inc., Fairfield, NJ). In the preferred embodiment, in which PHA is used as the mitogen, the cytokine profile for NCM has a profile of: CYTOCINE AMOUNT IL-1 10-200 pg / ml IL-2 100-500 units / ml IL-6 250-10,000 pg / ml IL-8 12,000-100,000 pg / ml IL-12 100-10,000 pg / ml IFN-gamma 50-15,000 pg / ml TNF-a 50-15,000 pg / ml CSF-G 50-1500 pg / ml CSF-GM 10-1500 pg / ml IL-3 / IL -4 / IL-7 Trace Amounts Immobilization of the mitogen produces a high performance of NCM than does the pulse techniques of the prior art. For example, the production of interleukins by a PHA pulse technique in serum-free media produced IL-2 at 0.20 units / ml medium (U.S. Patent Nos. 4,390,623 and 4,464,355). However, the present invention allows increased production with a pulse technique by adding a 4-aminoquinolone antibiotic to the serum-free media to hyperinduce interleukin producing IL-2 of about 8-140 units / ml. As predicted by the animal studies previously cited, this preparation characterized as a mixture of natural interleukin (NIM), at 200 units IL-2 / dose, increased the T lymphocyte counts in the blood of lymphopoenic patients with cancer in head and neck (Hadden et al., 1994), which had not been reported for rIL-2 at such a low dose. Similar effects of IL-2 have been reported only at doses greater than 5000 times the amount of IL-2 in NCM. Thus, it is important to note that the dose of equivalent IL-2 for NCM was used as an index of its potency and does not mean that it implies the total biological activity of NCM is that of only IL-2. In the preferred embodiment of the present invention, using continuous exposure for the mitogen by immobilization and the presence of the antibiotic 4-aminoquinolone, the NCM which is generated, generally contains IL-2 at 100-353 units / ml (an index of the power of preparation). In the less preferred embodiment, the invention can be practiced with the continued presence of the 4-aminoquinolone antibiotic and a pulse presence of the mitogen, producing NIM. This combination produces a higher cytokine level than the prior art with a pulse mitogen only, but does not produce the levels observed with the preferred embodiment of the present invention (NCM): continuous immobilized mitogen and 4-aminoquinolone antibiotic. The preferred embodiment in this situation is defined by the potency of the cytokine preparation which does not require the concentration resulting in loss of biological activity. The preferred modality at equivalent doses of IL-2 has the same biological activities as the less preferred modality (NIM or NI) (see Hadden et al., (1992) and the co-pending application by the same applicant filed on the same day as those present applications and assigned to the same assignee of the present invention). The production of mixtures of natural cytokines by lymphocytes stimulated with PHA is representative of plant lectins that have affinity for the T lymphocyte antigen receptor (TCR). Another such stimulant is concanavalin A (ConA). Similarly one might expect to induce high levels of interleukins under these conditions. The production of mixtures of natural cytokines by a monoclonal antibody such as OKT-3 which binds to TCR-related T cell surface receptors ie the CD3 complex, is representative of other monoclonal antibodies to such receptors such as DC2, CD28, CD45 and similarly would be expected to induce high levels of cytokines under these conditions. Other mitogens which stimulate B cells or on ocytes could be used in combination with mitogens, which stimulate T cells to provide a natural cytokine mixture. It would be expected that combinations of these stimulants would have additive effects as seen in Table III for IL-1 and IL-2. The observation contained herein that NCM produced by PHA is rich in IL-1, IL-2, IFN-gamma and also contains IL-12 (323 pg / ml), and has only trace amounts of IL-3, IL-4 and IL-7 indicate that under these conditions the PHA preferentially stimulates T-helper type I cells (TH-1) on type II T-helper cells. This allows the determination that these lymphokine preparations will have adjuvant activity to increase cellular immune responses (see Hadden, 1994). The production of NCM with immobilized mitogens can be used with the subsets of T cells (CD4, CD8, CD30, TH-1, TH-2, etc.) using the appropriate plates for the separation of cells by expansion before stimulation with PHA or other mitogens to obtain NCMs that are enriched for specific cytokines for these cell types. The NCM is formed in aliquots and stored at 4 ° C or less to maintain biological activity.

An effective amount of a natural cytokine mixture according to the protocol of the invention and as set forth in the co-pending application by the same applicant, filed on the same day as this application and assigned to the same assignee as this application and incorporated in the present for reference. The natural cytokine mixture can be administered to a mammalian host, preferably a human host and will have a specific cytokine profile and will generally have about 200-500 Units per dose of IL-2 for humans. Patients to receive treatment will be those with cellular immune deficiencies diagnosed either by themselves or in combination with other disease states. The function of the patient's T cells and blood levels will be evaluated as known in the art and if they are below normal, they will be a candidate for treatment since they have a cellular immune deficiency with the present invention designed to treat specifically the T cell abnormality. It is important to note that T cell lymphocytopenia is not only a reflection of cellular immune deficiency in the disease such as cancer and AIDS, but also a predictor of mortality in elderly men without disease (Bender et al., 1986).

The NCM is administered at low doses (200-500 units) of equivalence of IL-2 in accordance with good medical practice, taking into account the clinical condition of the individual patient, the site and method of administration, administration program and other factors known to doctors. The "effective amount" for the purposes of the present is thus determined by such considerations as are known in the art. It is important not to use high doses (> 1000 units / dose) since the effect is lost and the toxicity increases. The amount should be effective to show improvement in immune function in 25% of treated patients including, but not limited to, improved responses in measurements of cellular immune function, increased T lymphocyte levels in vivo, response of the improved skin test to remember antigens or NCM, improved survival rate, faster recovery or improvement or elimination of symptoms and in cancer reduction of the tumor mass. The NCM can be used with other treatments to improve immune function and treat cancer. The example of clinical use of NCM or NIM is exemplified by Hadden et al., (1994) in head and neck cancer. In the method of the present invention, the NCM can be administered in several ways. It should be noted that the NCM can be administered alone or in combination with pharmaceutically acceptable carriers. These can be administered subcutaneously or parenterally including intravenous, intraarterial, intramuscular, intraperitonal, perilymphatic, intralymphatic and intranasal administration. Site-specific administration is preferred, if possible. Implants and infusions of the compounds are also useful. The guidance is provided by the reference by Hadden et al., (1980) and Hadden et al. , (1994). For parenteral administration in humans, the present invention will generally be formulated in injectable dosage unit form, preferably in a pharmaceutically acceptable carrier medium and in a preferred embodiment it may be X-vivo-10 medium. Suitable carrier media include, but are not limited to, saline, squalene, dextrose solution, normal serum albumin, Ringer's solution 10, and the like. Optionally, secondary amounts of additives such as, for example, stabilizers, preservatives or buffers may be included in such a vehicle. Such a formulation is suitable for reconstruction in aqueous injections for parenteral administration. The mixture will typically be formulated in the carrier medium at a concentration of approximately 50 to 500 units of IL-2 (equivalence) / ml., preferably from about 150 to 350 units of IL-2 (equivalence) / ml. In addition, the mixture will have a consistent profile for other cytokines as established by the present invention. Optionally, the NCM can be carried in a lyophilized, stable, sterile formulation in which the active ingredients are mixed with a water soluble carrier and optionally, the stabilizer or without toxic preservatives. The various additives which increase the stability, sterility and isotonicity of the compositions, including antimicrobial preservatives, antioxidants, chelating agents and buffers can be added. The prevention of the action of microorganisms can be ensured by the presence of ciprofloxacin and different antibacterial and antifungal agents, for example parabens, chlorobutanol, phenol, sorbic acid and the like. In many cases, it will be advantageous to include isotonic agents for example sugars, sodium chloride and the like. Prolonged absorption of the injectable pharmaceutical form can be carried by the user of agents that retard absorption, for example aluminum monostearate and gelatin. In accordance with the present invention, however, any carrier, diluent, additive or delivery vehicle used would have to be compatible with the NCM and not alter the biological activity of the present invention. The dose and dosage regimen will depend mainly on the individual patient being treated, the patient's history, the type and magnitude of the biological damage to the patient, the duration of the treatment and the treatment protocol. The doses may be a single dose or multiple doses over a period of several days. The most preferred doses are those which achieve the maximum regression of the disease in the case of cancer or maximum reduction of symptoms in other diseases. It is noted that in humans they are generally treated longer than the mice exemplified herein, whose treatment has a duration proportional to the duration of the disease process and effectiveness of the drug. A pharmacological formulation of the NCM can be administered to the patient in an injectable formulation containing any compatible carrier, such as various carriers, adjuvants, additives and diluents; or the compounds used in the present invention can be administered parenterally to the patient in the form of subcutaneous slow release implants or targeting delivery systems such as infusion pumps, polymer matrices, liposomes and microspheres. An implant suitable for use in the present invention can take the form of a tablet which dissolves slowly after being implanted or a biocompatible delivery module well known to those skilled in the art. Such well-known dosage forms and modules are designed in such a way that the active ingredients are released slowly over a period of several days to several weeks. For example, such slow release form in infusion delivery systems will be contemplated to be employed in lung and esophageal cancer to deliver the MTN of the present described to regional nymphatic nodules in the vicinity of the cancer. Other cancers would use similar regional delivery techniques. Examples of well-known implants and modules useful in the present invention include: U.S. Patent No. 4,487,603, which discloses an implantable microinfusion pump for delivering medication at a controlled rate; U.S. Patent No. 4,486,194, which describes a therapeutic device for administering drugs through the skin; U.S. Patent No. 4,447,233, which discloses a medicament infusion pump for delivering the drug at an accurate infusion rate; U.S. Patent No. 4,447,224, which discloses an implantable variable flow infusion apparatus for the delivery of a continuous drug; U.S. Patent No. 4,439,196, which describes an osmotic drug delivery system having multiple chamber compartments; and U.S. Patent No. 4,475,196, which describes an osmotic drug delivery system. These patents are incorporated herein by reference. Many other such implants, delivery system and modules are well known to those skilled in the art. The present invention is effective to increase the numbers and function of thymocytes and for secondary secondary immunodeficiency as occurs in cancer, HIV infection, aging, etc. The method can be used to treat patients on an inpatient basis as well as an external patient, the latter being preferable. The above discussion provides a real basis for the preparation of a natural cytokine mixture (NCM). The methods used with and the utility of the present invention can be shown by the following examples. The examples demonstrate the utility of the invention in the murine system; however, equivalent preparations (NI and NIM) have shown activity in the human immune system (Pulley et al., 1994, Hadden et al., 1994). The mouse system was chosen because in addition to man, the mouse is the most studied species by structure and function of the immune system and is accepted by those with skill in the technique since it is highly predictive of the human response. So far only secondary differences have been observed between mice and man. Most of the mechanisms by which the mouse defends itself against several pathogens and tumors are essentially the same as for man. Mouse models have been used extensively in the evaluation of immunoregulators for use in humans. (Talmadge et al., 1985). Due to this prior technique, results from current murine experiments are predictors of human responses. Three examples demonstrate the predictive nature of the murine system with immunoregulators. Using a broad spectrum of murine tumor models the antitumor activity of interferons (IFN) has been shown (Borden, 1979; Talmadge et al., 1985) and correspondingly in humans, IFNs have shown activity against a large variety of tumors (Goldstein and Laslo, 1988). In a second example, using murine tumor models, there was no effect of levamisole used only on tumors, but activity was observed after chemotherapy (Symoens and Rosenthal, 1977; Spreafico, 1980). Similarly in humans, levamisole showed activity in human colon cancer, when it was used with 5-fluorouracil, but not only (Mutch and Hutson, 1991).

In a third example, using models of murine tumors the low dose of interleukin 2 (IL-2) showed to have antitumor activity without toxicity, while the high dose of IL-2 had activity especially with killer cells (LAK) activated by lymphokine (Rosenberg et al., 1985), but with potentially lethal toxicity. Human studies also showed the effectiveness of high-dose IL-2 ± LAK cells in malignant melanoma and renal cell cancer but with greater toxicity (Rosenberg, 1994). Even so, it is authorized by the FDA for renal cell cancer. Recent studies show the effectiveness of low-dose IL-2 in human cancer without toxicity (Cortesina et al., 1988 and 1994). The mechanisms are similar to the effect of the low dose observed in the murine system (Chirigos and Talmadge, 1985). The three previous examples of immunoregulators are now approved for clinical use in cancer and were highly predictive of studies of murine tumors. In addition, animal studies have shown mixing effects of natural interleukin (nIL) without sharing by recombinant interleukins (rlL). NIL, but not rIL-2, are active to restore and promote immune responses dependent on the thymus (Hadden, et al., 1992) and to promote resistance to malignant melanoma with cyclophosphamide (Kameda, et al., 1992). This same pattern has been observed in humans in which natural ILs were active in head and neck cancer in humans in a way not shared by recombinant IL-2. (Cortesina et al., 1988 and 1994, Hadden, et al., 1994, Mattijissen et al., 1991). EXAMPLES General Methods: All stages that relate to cell culture were performed under sterile conditions. General methods of cellular immunology not described herein, were performed as described in the general references for cellular immunology techniques such as Mishell and Shiigi (Selected Methods in Cellular Immunology, 1981) and as are known in the art. Materials Recombinant human beta 1 interleukin (rIL-1 beta) was a donation from Dr. C. Reynolds, Biological Response Modifiers Program, NCI (Frederick, MD). Human interleukin-2 (IL-2, specific activity of 640 U / ml) was obtained from Pharmacia AB (Silver Spring, MD). Recombinant IL-2 was a gift from G. Caspritz (Hoescht Pharm., Frankfort, Germany). Ciprofloxacin was purchased from Miles Inc., (West Haven, CT); The ofloxacin from McNeil Pharmaceutical (Spring House, PA); and the norfloxacin from Merck & Co (West Point, PA). Human serum albumin (HSA) was obtained from Armor Pharmaceuticals (Kankakee, IL). X-live media was purchased from Whittaker Bioproducts (Walkersville, MD). The 21-hydrocortisone hemisuccinate and ConA were purchased from Sigma Chemicals (St. Louis, MO). The PHA (HA-16) was obtained from Murex Diagnostics Ltd., Dartford, U.K.). The 0KT3 was purchased from Ortho Pharmaceuticals (Raritan, NJ). Preparation of natural cytokine preparations (NCM) White cells coated with inflammatory membrane of human blood from donors negative to hepatitis virus, negative to multiple HIV were collected. In an alternative embodiment, animals can be the cell source for veterinary uses. Donor cells are pooled and layered in Ficoll hypaque gradients (Pharmacia) to produce free lymphocytes of neutrophils and erythrocytes. (U.S. Patent Nos. 4,390,623 and 4,448,879). The alternative methods can be used in such a way that it would result in the same population start of lymphocytes as is known in the art. The lymphocytes are washed and distributed in V vivo-10 media (Whittaker Bioproducts) to flasks (MicroCELLector ™ T-25 Cell Culture Flasks) in which they are immobilized by stimulants, ie mitogens. In an established mode of experiments, the X vivo-15 and X vivo-20 media have been used as indicated. The immobilization process for the stimulants is as described by the manufacturer to immobilize several substances for expansion procedures, that is, separation of cells in the flasks. The cells are incubated for 24-48 hours in Live-10 X media with 80 μg / ml ciprofloxacin (Miles Lab) at 37 ° C in a CO 2 / air incubator. Alternatively, RPMI 1640 media can be used (Webb et al., 1973). Generally, HSA is used from 0.1 to 0.5% (weight by volume). After incubation, the supernatants are emptied and collected. Human serum albumin (HSA) can be added to stabilize the interleukins. The supernatants are stored at 4 ° C to -70 ° C. Characterization of the Supernatants The pooled supernatants are characterized by measuring the cytokine content by bioassay for IL-2 and the ELISA for the remaining interleukins IL-1, IL-15, CSF, TNF and IFN. Sterility is tested by culture in thioglycolate broth and the endotoxin is measured by the Limulus lysate assay as is known in the art. Standardization of supernatants for cytokine content Each supernatant is standardized either by concentration or amount administered, so that comparisons can be made.

Elimination of contaminants for supernatants DNA and virus exclusion, if used, will employ such techniques as ultrafiltration, column chromatography, sunflower, fractionation with ethanol, precipitation with polyethylene glycol / bentonite, gamma irradiation and / or solvent treatment. detergent as used for intravenous gamma globulin and monoclonal antibodies (eg IGIV News Update brochure). Model The model of thymic involution induced by hydrocortisone in old mice was used unless indicated otherwise (Hadden et al., 1992). Laboratory Animals Female BALB / c (Life Science, St. Petersburg, FL) old withdrawals from the breeding site of mice (8-9 months) whose scams have begun to regress were used in the in vivo tests. The mice are coupled by weight and meet randomly in groups of five. The animals were fed standard laboratory diets with water to drink ad libtum. All mice, with the exception of a control group, were treated intraperitoneally (ip) with hydrocortisone (5 mg / mouse in 0.1 ml of 0.9% sodium chloride) for two consecutive days to induce chemical thymectomy and reduction of spleen weight .

Adult mice treated with hydrocortisone oyster-rum acute thymic involution (less than 30% control) and reduction in spleen size (less than 80% control) at two days with progressive recovery at 10 days. Experimental Design Each treatment group had five (5) animals and each experiment was repeated 2-5 times. Treatment was initiated intraperitoneally (i.p.) on Day 3 and continued once a day for a total of five (5) days. The treatment groups were injected with one of the following treatments in vivo as indicated in the text: 1. pyrogen-free saline solution (controls); 2. recombinant interleukin-1 (rIL-1; 4ng); 3. Recombinant interleukin-2 (rIL-2, 50 units); 4. rIL-1 + rIL-2 (4ng + 50 units, respectively) 5. natural cytokine mixture (NCM, 50 units of equivalence of IL-2); On day 8, the mice were weighed, sacrificed by cervical dislocation and their spleens and thymuses removed and weighed. Organs were crushed, residual erythrocytes were lysed using ammonium chloride (Mishell and Shiigi, 1981), and cells were counted.

The proliferative response of the cells to various substances was then determined. A sample of cells was prepared for cell culture at 37 ° C, 5% C02 in medium RPMI 1640 with 5% fetal bovine serum, penicillin (100 U / ml), streptomycin (100 μg / ml) and 2-mercaptoethanol (2 x 10 ~ 5M). The cells were plated in 0.2 ml microwell plates per quadruplicate at a concentration of 1. 5 x 106 / ml and incubated for 72 hours with one of the following, as indicated in the text: 1. control diluent (complete RPMI 1640 medium); 2. rIL-1 (1 ng / ml); 3. rIL-2 (2 Units / ml); 4. NCM (2 Units / ml equivalence of IL-2) 5. concanavalin A (ConA, 1.5 μg / ml) 6. phytohe agglutinin (PHA, 0.5 μg / ml). To measure DNA synthesis, the culture is terminated with an 18-hour pulse of tritiated thymidine (3H-Thymidine, New England Nuclear, Boston, MA, specific activity of 6.7 Ci / mM), is harvested with an automatic, multiple sample harvester and processed for liquid scintillation counting. Marker studies were also performed as described by Hadden et al., (1992). The results are expressed as the arithmetic mean of cpm from three samples for each animal. To simplify the representation of the data obtained with different animals, the results are collected and calculated together and in some cases they are expressed as a relation for the control and others as means + brackets for the standard error (SEM). Statistical Analysis The Student's T test was used to analyze the data as appropriate. Example 1 The objective was to find a way to simulate lymphocytes to produce high concentrations of interleukin-2 in the absence of serum and in a form, which does not produce significant amounts of PHA in the supernatant. To do this, the PHA was immobilized on the surface of activated cell culture flasks for the selection of cell subseries (AIS microCELLector ™ T-25 plates) as described in the manufacturer's instructions to "extend" cell separation. The media used in these experiments were X vivo-10 (Whittaker) and is approved for administration to humans by the US Food and Drug Administration for killer cell (LAK) protocols activated with interleukin-2-lymphokine. Serum-free media capable of supporting the proliferation of human lymphocytes as minimal essential media (MEM) or RPMI-1640 (Sigma) can also be used.

Initial experiments indicated that the PHA (HA-16, Murex Diagnostics Ltd., Dartford, UK) ^ can be immobilized by the technique described by the manufacturer and that under appropriate optimal conditions the cell number of 7.5-15 xl06 / ml can be had, exposure time of 24 hours - 48 hours, and PHA concentration of 25 or 50 μg / ml a high yield of interleukin-2 in the supernatant without serum. The performance was superior to the previous methods (pulse technique) that uses brief exposures to PHA (NI) followed by washing and subsequent culture with ciprofloxacin (NIM) in medium without serum (Table 1). Therefore, this flask procedure is used to generate the mixture NCM. TABLE 1 IL content of the supernatant / ml Brief exposure to PHA (NI) 2-20 units Brief exposure to PHA and ciprofloxacin (NIM) (80 μg / ml) 8-140 units Immobilization to the flask with PHA and ciprofloxacin (80 μg / ml) 100-353 units The content of IL-2 is measured in the supernatant using the cell line dependent on IL-2 CTLL by the methods described by Gillis et al. (1978). IL-2 was quantified in international units against the known standard containing 640 units (Pharmacia AB). The cell-free supernatants from the flasks incubated with cells are tested on human lymphocytes to determine if residual PHA was present in sufficient amounts to produce a proliferative response. Any residual PHA greater than 0.01 μg / ml would give such a response. In the absence of the cells, small amounts of PHA were observed in the supernatant at 40-48 hours; however, when PHA (25 μg / ml) was used for only 24 hours, these concentrations were negligible. 24 hours of incubation were considered optimal. When the new flasks are compared with the old flasks under comparable conditions, a higher level of IL-2 was observed with the older flasks. Therefore, old flasks generally, but not always, were used in the examples to generate the NC mixture. Example 2 A comparison of live X-10, live X-15 and live X-20 (Whittaker) and MEM in the present invention, was carried out and shown in Figures 1-3. X vivo-10 and X vivo-15 are approved for administration to humans by the Food and Drug Administration of the United States for killer cell (LAK) protocols ^ activated by interleukin-2-lymphokine. The generation of NCM was compared in different media using continuous exposure against pulse exposure to PHA at 1 μg / ml (Figure 1). The effect of cell concentration was explored with continuous exposure to PHA at 1 μg / ml (Figure 2) and PHA at 2 μg / ml (Figure 3). The optimal combination of these factors was found to be the continuous exposure by immobilization in X-vivo-10 at cell concentrations of 2.5 or 5.0 xlO ^ / ml with PHA at 2 μg / ml or at 5xl06 cells / ml, with PHA at 1 μg / ml. Because the cell produced is more efficient at 2.5 x 10 6 cells / ml, the concentration with PHA at 2 μg / ml is chosen as the optimum. Example 3 Preliminary experiments in tubes instead of flasks were performed to determine the parameters for ciprofloxacin and two others for the antibiotics 4-aminoquinolone (Norfloxacin and Ofloxacin) to increase the production of cytokine from human lymphocytes after exposure to PHA. Table II shows that 80 μl / ml of each of these 4-aminoquinolone antibiotics increased the production of IL-1, IL-2, IL-6, IFN-6, TNFα, and G-CSF. The production of IL-8 was maximum. IL-3, IL-4 and IL-7 were undetectable under these circumstances in all supernatants. These results indicate that under these serum-free conditions all 4-aminoquinolones tested at 80 μg / ml increased PHA-induced cytokine production under serum-free conditions. TABLE II PHA Ciprofloxacin Norfloxacin Ofloxacin Sola and PHA and PHA and PHA IL-l-ß 81 1080 783 813 IL-2 ND 120 32 82 IL-6 1665 > 3000 > 3000 > 3000 IL-8 18000 > 18000 > 18000 > 18,000 IFN-gamma ND 750 210 380 TNF-Á 54 1935 1500 4000 GM-CSF 114 4. 5 4. 5 72 G-CSF 41 555 800 630 Units for cytokines other than IL-2 are pg / ml and for IL-2 international units / ml. ND: not detectable. Example 4 It was also determined that a monoclonal antibody, OKT-3, (Ortho), which induces T lymphocytes to proliferate and produce interleukins can be used as a stimulant under these conditions. Table III shows that OKT-3 induced the cytokines similar to those induced by PHA plus ciprofloxacin with cells incubated in flasks as set forth in Example 1, IL-3, 4 and 7 were not detected with any set of stimulants. OKT-3 produced a small additive effect for several ILs when binding with PHA and ciprofloxacin (CIPRO). TABLE III CIPRO OKT-3 + CIPRO OKT-3 + PHA + PHA IL-l-ß 1080 1530 1125 IL-2 120 340 ND IFN-gamma 750 4660 11280 IL-6 > 3000 > 3000 1980 IL-8 > 18000 > 18000 > 18000 TNFÁ 1935 2700 2500 GM-CSF 4.5 12 75 G-CSF 555 375 ND Interleukin units apart from IL-2 are in pg / ml and for IL-2 international units / ml. ND - not detectable. Example 5 To show the superiority of the NCM rIL-1 in vitro, splenocytes and mouse thymocytes were cultured with MEM and rIL-2 at comparable concentrations of IL2 as determined by the bioassay and DNA synthesis measured by thymidine incorporation. tritiated NCM induced greater proliferation of splenocytes (Figure 4) and thymocytes (Figure 5), then rIL-2 was based on the content of IL2. Example 6 In a series of experiments as set forth in Figures 6 and 7, mice with involuted thymuses were treated in vivo with rIL-1, rIL-2, combinations of these factors, NCM or saline (controls). Spleens and thymuses were removed, the cells tested for cell proliferation responses against interleukins (IL-1, IL-2), NCM and the mitogen ConA. The results are expressed as the ratio for the control treated with the saline solution. In vivo treatment with rILl, rIL-2 and its combination (rIL-1 and rIL-2) had no significant effect to increase the proliferative responses of splenocytes (Figure 6) or thymocytes (Figure 7) for in vitro stimulation with IL-1, IL-2 NCM or ConA. Treatment with NCM in vivo significantly increased both splenocytes and thymocytes for all four stimuli. These results are consistent with an increased sensitivity of these cells for stimulation and / or an increase in the number of sensitive cells. Example 7 Figures 8 and 9 demonstrate the effect of NCM treatment in vivo on markers of splenocytes and thymocytes. Non-mature T cells are indicated by - and may represent T lymphocyte precursors, particularly in the thymus. The-NCM proportionally increased this population in the spleen and thymus. Immature T cells are indicated by ++ and this population is proportionally decreased in the thymus by treatment with NCM. The mature T are indicated by CD4 + and CD8 +. The NCM increased the proportions of mature T cells in the thymus, but not in the spleen. These results are consistent with an effect of NCM to increase T cell precursors and to promote their development to mature T cells in the thymus. Example 8 Figures 10 and 11 demonstrate the responses of splenocytes and thymocytes in vitro to the media (RPMI), rIL-1 (IL-j, rIL-2 (IL2), or NCM after in vivo treatment with the media. control or NCM in the hydrocortisone model.The mice were treated as described above.These data demonstrate that the NCM increases the responses of splenocyte background, the splenocyte responses to IL-1 and IL-2, but not to NCM and background thymocyte responses and thymocyte responses to IL-1, IL-2, and NCM Figures 12 and 13 demonstrate the responses of splenocytes and thymocytes in vitro to ConA to PHA after in vivo treatment with the control means or NCM The mice were treated as described above.

In vitro studies demonstrate the superiority of NCM over rIL-2 at equivalent doses in splenocytes and thymocytes sensitized to proliferation signals. The effects on thymocytes reflect the promotion of differentiation as well. The composition of NCM, but not rIL-1, rIL-2, or its combination, potentially promotes T lymphocyte function in vivo (IL responses) and development (mitogen responses and cellular markers), which is therapeutically important in any of the therapeutic measures that require the stimulation of the immune system and restoration to a partial functioning of a damaged or defective immune system. For example, chemotherapeutic agents can damage cells, including T lymphocytes, involved in the immune response. The present invention by stimulating the functioning and development of the T lymphocytes can restore, either partially or completely, this characteristic of the immune system, if damaged. Throughout this application, various publications, including United States patents, are mentioned by appointment or number. All citations for publications are listed in the following. The descriptions of these publications and patents in their entireties are incorporated herein by reference in this application to more fully describe the state of the art to which this invention pertains. The invention has been described in an illustrative form and it should not be understood that the terminology, which has been used is intended to be in the nature of the words of the description rather than limitation. Obviously, many modifications and variations of the present invention are possible in light of the above teachings. Therefore, it should be understood that within the scope of the appended claims, the invention may be practiced in any other way than that specifically described. REFERENCES AIS Technical Bulletin No. 9003, "Covalent Immobilization of Protein-Reagent Polystyrene Antibodies". Belldegrun and Rosenberg, "Adoptive Immunotherapy of Urological Tumors", Cancer Treat. Res. 46: 213-233, 1989 Bender et al., "Absolute Peripheral Blood Lymphocyte Count and Subsequent Mortality of Elderly Men" JAGS 34: 649-654, 1986 BioWhi ttaker Catalog, "Adoptive Immunotherapy and Therapy Genetics "Borden," Interferons: Fundamental Reason for Testing Clinics in Neoplastic Disease ", Ann. Int. Med. 91: 492-479, 1979.

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Claims (15)

1. CLAIMS 1. A method for producing a natural cytokine mixture, characterized in that it includes the steps of immobilizing at least one mitogen in a tissue culture vessel, suspending an isolated population of neutrophil-free lymphocytes and erythrocytes in serum-free media, the lymphocytes suspended in the container, cultivate the lymphocytes, remove the media, and characterize the means for the production of the cytokines.
2. 2. The method according to claim 1, characterized in that the serum-free media are selected from X-vivo-10 and X-vivo-15.
3. 3. The method according to claim 1, characterized in that the mitogen is selected from the group consisting of lectins and monoclonal antibodies that stimulate the lymphocytes to produce cytokines.
4. 4. The method according to claim 3, characterized in that the lectins are selected from the group consisting of phytohemagglutinin (PHA) and concanavalin A (ConA).
5. 5. The method in accordance with the claim 3, characterized in that the monoclonal antibodies are selected from the group consisting of 0KT3, anti-CD2, anti-CD28, and anti-CD45.
6. 6. The method of compliance with the claim 3, characterized in that a combination of mitogens was used.
7. 7. The method of compliance with the claim 4, characterized in that the mitogen is PHA.
8. 8. The method of compliance with the claim 1, characterized in that the cytokines to be characterized include at least one of the group consisting of INF-gamma, IL-1, IL-2, IL-3, IL-4, IL-6, IL-7, IL -8, GM-CSF, G-CSF and TNF-Qf.
9. 9. The method of compliance with the claim 1, characterized in that the serum-free media contains a 4-aminoquinolone antibiotic.
10. The method according to claim 9, characterized in that the antibiotic 4-aminoquinolone is selected from the group consisting of Ciprofloxacin, Norfloxacin and Ofloxacin.
11. The method according to claim 1, characterized in that the tissue culture vessel is AIS microCELLector ™ T-25 flasks for culturing activated cells on the surface.
12. 12. The method according to claim 1, characterized in that the culture is for twenty-four hours to forty-eight hours.
13. 13. A natural mixture of cytokines produced by culturing lymphocytes in the presence of an immobilized mitogen and media without serum.
14. 14. The natural mixture of cytokines according to claim 13, characterized in that the serum-free media contains a 4-aminoquinolone antibiotic.
15. 15. The natural cytokine mixture according to claim 13, characterized in that the mitogen is phytohemagglutinin and that it contains a cytokine profile of IL-1 at 10-2000 pg / ml, IL-2 at 100-500 units / ml, IL-6 at 250-10,000 pg / ml, IL-8 at 12,000-100,000 pg / ml, IL-12 at 100-10,000 pg / ml, IFN-gamma at 50-15,000 pg / ml, TNF-a at 50- 15,000 pg / ml, CSF-G at 50-1500 pg / ml, CSF-GM at 10-1500 pg / ml, and IL-3, IL-4, IL-7 present in trace amounts.