MX2008003912A - Method for enhancing immune responses in mammals - Google Patents

Abstract

Provided is a method for enhancing an immune response in a mammal to facilitate the elimination of a chronic pathology. The method involves the removal of immune system inhibitors such as soluble TNF receptor from the circulation of the mammal, thus, enabling a more vigorous immune response to the pathogenic agent. The removal of immune system inhibitors is accomplished by contacting biological fluids of a mammal with one or more binding partners such as TNFαmuteins capable of binding to and, thus, depleting the targeted immune system inhibitors from the biological fluids. Particularly useful is an adsorbent matrix composed of an inert, biocompatible substrate joined covalently to a binding partner, such as a TNFαmutein, capable of specifically binding to a targeted immune system inhibitor such as soluble TNF receptor.

Description

METHOD TO IMPROVE IMMUNE RESPONSES IN MAMMALS FIELD OF THE INVENTION This invention relates generally to the field of immunotherapy and, more specifically, to methods for improving the immune responses of the hosts.

BACKGROUND OF THE INVENTION The immune system of mammals has evolved to protect hosts against the growth and proliferation of potentialy harmful agents. These agents include infectious microorganisms such as bacteria, viruses, fungi and parasites which exist in the environment and which, at the time of introduction into the host body, can induce various pathological conditions. Other pathological conditions can be derived from agents not acquired through the environment, but rather arise spontaneously within the body of the host. The best examples are the numerous malignancies that are known to occur in mammals. Ideally, the presence of these harmful agents in a host activates the mobilization of the immune system to effect the destruction of the agent and thus restore the inviolability of the host environment.

The destruction of pathogenic agents by the immune system involves a variety of effector mechanisms which can be grouped generally into two categories: innate and specific immune. The first line of defense is mediated by the innate immune mechanisms. Innate immunity does not discriminate among the multitude of agents that may enter the host body. Rather, it responds in a generalized manner that employs the inflammatory response, phagocytes and plasma-transmitted components such as complement and interferons. In contrast, specific immunity does discriminate among pathogenic agents. Specific immunity is mediated by B and T lymphocytes and serves, in large part, to amplify and focus innate immune effector mechanisms.

The elaboration of an effective immune response requires contributions from both the innate and specific immune mechanisms. The function of each of these branches of the immune system individually, as well as their interaction with one another, is carefully coordinated, both temporally / spatially and in terms of the particular cell types involved. This coordination results from the actions of a number of soluble immuno-stimulatory mediators or "immune system stimulators" (reviewed in, Trinchieri et al., J. Cell, Biochem 53: 301-308 (1993)). Some of these stimulators of the immune system initiate and perpetuate the inflammatory response and the systemic sequelae in waiting. Examples of these include, but are not limited to, the pro-inflammatory mediators of factors a and ß of tumor necrosis, interleukin-1, interleukin-6, interleukin-8, interferon-y and the RANTES chemokines, macrophage inflammatory proteins 1 -ay 1-ß and the macrophage and activation factor. Other stimulators of the immune system facilitate the interactions between B and T lymphocytes of specific immunity. Examples of these include, but are not limited to, interleukin-2, interleukin-4, interleukin-5, interleukin-6 and interferon-y. Still other stimulators of the immune system mediate bidirectional communication between specific immunity and innate immunity. Examples of these include, but are not limited to, interferon-7, interleukin-1, a and ß factors of tumor necrosis and interleukin-12. All these stimulators of the immune system exert their effects by binding specific receptors on the surface of host cells, resulting in the delivery of intracellular signals that alter the function of the target cell. Cooperatively, these mediators stimulate the activation and proliferation of immune cells, recruit them into particular anatomical sites and allow their collaboration in the elimination of offensive agents. The immune response induced in any individual is determined by the particular complement of the stimulators of the immune system produced and by the relative abundance of each.

In contrast to the immune system stimulators described above, the immune system has evolved other soluble mediators that serve to inhibit immune responses (reviewed in Arend, Adv. Int. Med. 40: 365-394 (1995)). These "immune system inhibitors" They provide the immune system with the sensitivity to terminate responses in order to prevent the establishment of a chronic inflammatory state with the potential to damage the host tissue. The regulation of host immune function by inhibitors of the immune system is achieved through a variety of mechanisms as described above.

First, certain inhibitors of the immune system bind directly to the stimulators of the immune system and thus prevent them from binding to the plasma membrane receptors in the host cells. Examples of these types of immune system inhibitors include, but are not limited to, the soluble receptors for a and b factors of tumor necrosis, interferon-y, interleukin-1, interleukin-2, interleukin-4, interleukin-6. and interleukin-7.

Second, certain inhibitors of the immune system antagonize the binding of the stimulators of the immune system to its receptors. By way of example, the interleukin-1 receptor antagonist is known to bind to the interleukin-1 membrane receptor. It does not deliver activation signals to the target cell but, because it occupies the interleukin-1 membrane receptor, it blocks the effects of interleukin-1.

Third, particular inhibitors of the immune system exert their effects by binding receptors on host cells and signaling a decrease in their production of stimulators of the immune system. Examples include, but are not limited to, interferon-β, which decreases the production of two key pro-inflammatory mediators, tumor necrosis factor a and interleukin-1 (Coclet-Ninin et al., Eur. Cvtokine Network 8: 345-349 (1997)) and interleukin-10, which suppresses the development of cell-mediated immune responses by inhibiting the production of the immune system stimulator, interleukin-12 (D'Andrea et al., J. Exp. Med. 178: 1041-1048 (1993)). In addition to decreasing the production of stimulators of the immune system, certain inhibitors of the immune system also improve the production of other inhibitors of the immune system. By way of example, interferon-a26 inhibits interleukin-1 and the production of tumor necrosis factor-a and increases the production of corresponding inhibitors of the immune system, antagonist of the interleukin-1 receptor and soluble receptors for tumor necrosis factors a and β (Dinarello, Sem. in Oncol 24 (3 Suppl 9): 81-93 (1997).

Fourth, certain inhibitors of the immune system act directly on immune cells, inhibiting their proliferation and function, thus decreasing the vigor of the immune response. By way of example, the growth transforming factor-β inhibits a variety of immune cells and significantly limits inflammation and cell-mediated immune responses (reviewed in Letterio and Roberts, Ann. Rev. Immunol. 16: 137-161 ( 1998)). Collectively, these various immunorepressive mechanisms are intended to regulate the immune response, both quantitatively and qualitatively, to minimize the potential for collateral damage to the host's own tissue.

In addition to the inhibitors produced by the host immune system for self-regulation, other inhibitors of the immune system are produced by infectious microorganisms. For example, many viruses produce molecules which are viral homologs of the host immune system inhibitors (reviewed in Spriggs, Ann, Rev. Immunol., 14: 101-130 (1996)). These include homologues of host complement inhibitors, interleukin-10 and soluble receptors for interleukin-1, factors a and β of tumor necrosis and interferons a, β and y. Similarly, helminthic parasites produce homologs of the host immune system inhibitors (reviewed in Riffkin et al., Immunol Cell Biol. 74: 564-574 (1996)) and various bacterial genera are known to produce immunosuppressive products (reviewed in Reimann et al., Scand, J. Immunol., 31: 543-546 (1990)). All these inhibitors of the immune system serve to suppress the immune response during the initial stages of infection, to provide an advantage to the microbe and to improve the virulence and chronicity of the infection.

A role for inhibitors of the immune system derived from the host in chronic diseases has also been established. In most cases, this reflects a polarized t-cell response during the initial infection, where the production of mediators immunosuppressive (ie, interleukin-4, interleukin-10 and / or transforming growth factor-β) dominates over the production of immunostimulatory mediators (ie, interleukin-2, interferon-y and / or tumor necrosis factor β) ) (reviewed in Lucey et al., Clin. Micro. Rev. 9: 532-562 (1996)). The overproduction of immunosuppressive mediators of this type has been shown to produce chronic, non-curative pathologies in a number of medically important diseases. These include, but are not limited to, diseases resulting from infection by: 1) Plasmodium falciparum parasites (Sarthou et al., Infect Immun .. 65: 3271-3276 (1997)), Trypanosoma cruzi (reviewed in Laucella et al. ., Argentine Journal of Microbiology 28: 99-109 (1996)), Leishmania major (reviewed in Etges and Muller, J. Mol. Med. 76: 372-390 (1998)) and certain helminthiases (Riffkin et al., Supra). ); 2) intracellular bacteria, Mycobacterium tuberculosis (Baliko et al., FEMS Immunol.Med., Micro 22: 199-204 (1998)), Mycobacterium avium (Bermudez and Champsi, Infect.Immun.61: 3093-3097 (1993) ), Mycobacterium Leprae (Sieling et al., J. Immunol., 150: 5501-5510 (1993)), Mycobacterium bovis (Kaufmann et al., Ciba Fdn. Symp. 195: 123-132 (1995)), Brucella abortus ( Fernandes and Baldwin, Infect.Immun 63: 1 130-1133 (1995)) and Listeria monocytogenes (Blauer et al., J. Interferon Cvtokine Res. 15: 105-114 (1995)) and 3) intracellular fungi, Candida albicans (reviewed in Romani et al., Immunol Res 14: 148-162 (1995)). The inability to spontaneously resolve the infection is also influenced by other inhibitors of the immune system derived from the host. By way of example, the interleukin-1 receptor antagonist and soluble receptors for tumor necrosis factors a and b are produced in response to the production of interleukin-1 and soluble receptors for a and / or b-factors of necrosis of tumor driven by the presence of numerous infectious agents. Examples include, but are not limited to, Plasmodium falciparum infections (Jakobsen et al., Infect.Immun.66: 1654-1659 (1998), Sarthou et al., Supra), Mycobacterium tuberculosis (Balcewicz-Sablinska et al., J. Immunol., 161: 2636-2641 (1998)) and Mycobacterium avium (Eriks and Emerson, Infec. Immun 65: 2100-2106 (1997)). In cases where the production of any of the aforementioned immune system inhibitors, either individually or in combination, terminates or otherwise alters the ability of the immune response before elimination of the pathogenic agent, a chronic infection may result.

In addition to this role in infectious diseases, inhibitors of the immune system derived from the host also contribute to chronic malignancies. Studies provide convincing evidence for the receptor for soluble tumor necrosis factor I (sTNFRI) in patients with cancer. The nanomolar concentrations of sTNFRI are synthesized by a variety of activated immune cells in patients with cancer and, in many cases, by the tumors themselves (Aderka et al., Cancer Res. 51: 5602-5607 (1991), Adolf and Apfler, J. Immunol., Meth. 143: 127-136 (1991)). In addition, the levels of sTNFRI that are circulating are often significantly elevated in patients with cancer (Aderka et al., Supra, Kalmanti et al., Int. J. Hematol., 57: 147-152 (1993), Elsasser- Beile et al., Tumor Biol. 15: 17-24 (1994), Gadducci et al., Anticancer Res. 16: 3125-3128 (1996), Digel et al., J. Clin. Invest. 89: 1690-1693 (1992)), decline during remission and increase during the advanced stages of tumor development (Aderka et al., Supra, Kalmanti et al., Supra, Elsasser-Beile et al., Supra, Gadducci et al., Supra) and, when present at high levels, they correlate with poorer treatment outcomes (Aderka et al., supra). These observations suggest that sTNFRI helps tumor survival by inhibiting anti-tumor immune mechanisms which employ tumor necrosis factor A and / or β (TNF) and argue favorably for the clinical manipulation of sTNFRI levels as a therapeutic strategy against cancer.

Direct evidence that the elimination of immune system inhibitors provides clinical benefits is derived from the evaluation of Ultrapheresis, a promising experimental therapy against cancer (Lentz, J. Biol. Response Modif. 8:51 -527 (1989), Lentz, Ther.Apheresis 3: 40-49 (1999), Lentz Jpn. J. Apheresis 16: 107-1 14 (1997)). Ultrapheresis involves the extracorporeal fractionation of the plasma components by ultrafiltration. Ultrapheresis selectively removes plasma components within a defined molecular size range and has been shown to provide significant clinical advantages for patients presenting a variety of tumor types. Ultrapheresis induces pronounced inflammation at tumor sites, often less than an hour after initiation. This rapidity suggests a role for cellular mediators and / or preformed chemists in the elaboration of this response inflammatory and reflects the elimination of plasma inhibitors that occur naturally from that response. In fact, inhibitors of the immune system of TNF a and β, interleukin-1 and interleukin-6 are eliminated by Ultrapheresis (Lentz, Ther .. Apheresis 3: 40-49 (1999)). Notably, the removal of sTNFRI has been correlated with the observed clinical responses (Lentz, Ther.Aphresis 3: 40-49 (1999), Lentz, Jpn. J. Apheresis 16: 107-1 14 (1997)).

Ultrapheresis is in direct contrast to more traditional approaches which have tried to stimulate immunity through the addition of stimulators of the immune system. Above these has been the infusion of supraphysiological levels of TNF (Sidhu and Bollón, Pharmacol Ther 57: 79-128 (1993)) and interleukin-2 (Maas et al., Cancer Immunol. Immunother. 141-148 (1993)), which indirectly stimulates the production of TNF. These therapies have enjoyed limited success (Sidhu and Bollón, supra, Maas et al., Supra) because: 1) the levels used were extremely toxic and 2) each one increases the plasma levels of the immune system inhibitor , sTNFRI (Lantz et al., Cvtokine 2: 402-406 (1990), Miles et al., Brit. J. Cancer 66: 1195-1199 (1992)). Taken together, these observations support the utility of Ultrapheresis as a biotherapeutic approach to cancer - one that involves the removal of inhibitors of the immune system, rather than the addition of stimulators of the immune system.

Although Ultrapheresis provides advantages over traditional therapeutic approaches, there are certain drawbacks that limit its clinical utility. Not only immune system inhibitors are eliminated by Ultrapheresis, but other plasma components, including beneficial ones, are eliminated since the discrimination between the removed and retained plasma components is based only on molecular size. A further drawback of Ultrapheresis is the significant loss of circulatory volume during treatment, which must be compensated for by the infusion of replacement fluid. The most effective replacement fluid is an ultrafiltrate produced, in an identical manner, from plasma from donors that do not carry a tumor. A typical treatment regimen (15 treatments, each with the elimination of approximately 7 liters of ultrafiltrate) requires more than 200 liters of donor plasma for the production of the replacement fluid. The chronic shortage of donor plasma, combined with the risks of infection by human immunodeficiency virus, hepatitis A, B or C or other etiological agents, represents a severe impediment to the broad implementation of Ultrapheresis.

Due to the beneficial effects associated with the elimination of immune system inhibitors, there is a need for methods which can be used to specifically strip those inhibitors from circulation. Such methods should ideally be specific and not remove other circulatory components and should not result in any significant loss of circulatory volume. The present invention satisfies these needs and also provides related advantages.

SUMMARY OF THE INVENTION A method is provided for stimulating immune responses in a mammal through stripping of immune system inhibitors such as soluble TNF receptors present in the circulation of the mammal. The stripping of immune system inhibitors such as soluble TNF receptors can be effected by removing the biological fluids from the mammal and contacting these biological fluids with a binding partner, eg, mutein TNFa, capable of selectively binding to the target inhibitor of the host. immune system.

Binding couples useful in these methods include TNFα muteins that have specificity for soluble TNF receptors. In addition, mixtures of TNFα muteins having specificity for one or more soluble TNF receptors can be used.

As an example, a binding partner, such as a TNFα mutein, can be previously immobilized on a solid support to create an "absorbent matrix" (Figure 1). Exposure of biological fluids to said absorbent matrix will allow binding by the inhibitor of the immune system such as a soluble TNF receptor thus effecting a decrease in its abundance in biological fluids. The treated biological fluid can be returned to the patient. The total volume of biological fluid to be treated and the treatment speed are individualized parameters for each patient, guided by the induction of vigorous immune responses while minimizing toxicity. The solid support (i.e., inert medium) may be composed of any material useful for that purpose, including, for example, hollow fibers, cellulose-based fibers, synthetic fibers, flat or pleated membranes, silica-based particles, beads macroporous and similar.

As another example, the binding partner such as mutein TNFa can be mixed with the biological fluid in a "stirred reactor" (Figure 2). The binding partner-inhibitor complex of the immune system can then be removed by mechanical, chemical or biological means or methods and the altered biological fluid can be returned to the patient.

Conjugates comprising a tumor necrosis factor a (TNF a) mutein fixed to a substrate are also provided.

In addition, apparatuses incorporating either the absorbent matrix or the stirred reactor are provided.

BRIEF DESCRIPTION OF THE DRAWINGS The accompanying drawings, which are incorporated in, and constitute a part of this specification, illustrate various aspects of the invention and together with the description, serve to explain the principles of the invention.

Figure 1 illustrates schematically an "absorbent matrix" configuration of one aspect of the compositions, conjugates and methods disclosed, wherein a) blood pressure, b) positive displacement blood pump, c) blood separation, d) blood cells, blood, e) acellular fraction, f) immobilized binding partner, g) stripped acellular fraction and h) pump volumetric In this example, the blood is removed from the patient and separated into a cellular and acellular component, or fractions thereof. The acellular component, or fractions thereof, is exposed to the absorbent matrix to effect the binding and thus stripping of an objective inhibitor of the immune system such as a tumor necrosis factor (TNF) receptor. The altered acellular component, or fractions thereof, is then returned to the patient contemporaneously.

Figure 2 schematically illustrates a "stirred reactor" configuration of one aspect of the compositions, conjugates and methods disclosed, wherein a) blood pressure, b) positive displacement blood pump, c) blood separation, d) blood cells, blood, e) acellular fraction, f) inhibitor, g) binding partner, h) volumetric pump ei) withdrawal of complex binding partner / inhibitor. In this example, the blood is removed from the patient and separated into a cellular and acellular component, or fractions thereof. A binding partner such as a TNF mutein is added to the acellular component, or fractions thereof. Subsequently, the complex binding partner (mutein TNFa) / inhibitor of the immune system (soluble TNF receptor) is removed by mechanical, chemical or biological means or methods of the acellular component, or fractions thereof and the altered fluid is returned to the patient contemporaneously.

. Figure 3A shows an alignment of TNFa sequences from various mammalian species (mouse, SEQ ID NO: 10, rat, SEQ ID NO: 11, rabbit, SEQ ID NO: 12, cat, SEQ ID NO: 13, dog, SEQ ID NO: 14, sheep, SEQ ID NO: 15, goat, SEQ ID NO: 16, horse, SEQ ID NO: 17, cow, SEQ ID NO: 18, pig, SEQ ID NO: 19, human, SEQ ID -NO: 2). The upper sequence shows the conserved amino acids through the species shown (SEQ ID NO: 1) (fully conserved or with one exception). The non-conserved amino acids are indicated by "." (taken from Van Ostade et al., Prot. Ena 7: 5-22 (1994), which is incorporated herein by reference). Figure 3B shows an alignment of a conserved TNFα sequence with human TNFα and six representative TNFα muteins, designated mutein 1 (SEQ ID NO: 3), mutein 2 (SEQ ID NO: 4), mutein 3 (SEQ ID NO: 5) , muteina 4 (SEQ ID NO: 6), mutein 5 (SEQ ID NO: 7) and mutein 6 (SEQ ID NO: 8). The four muteins differ from the human sequence by simple amino acid substitutions, indicated in bold and underlined. Figure 3C shows the representative consensus TNFa sequence (SEQ ID NO: 9).

Figure 4 shows the presence of human TNFα and TNFα muteins 1, 2, 3 and 4 in periplasmic preparations of Escherichia coli transformed with the respective expression constructs, wherein a) relative TNF, b) periplasm, c) TNF, d) M1, e) 2, f) M3 and g) 4.

Figure 5 shows that the TNFa muteins bind to sTNFRI. The wells of a microtiter plate were covered with TNFa, blocked and incubated with sTNFRI either in the presence or absence of inhibitors, TNFa and TNFa muteins 1, 2 and 4, where a) relative binder, b) concentration of inhibitor ( ug / ml), c) none, d) TNF, e) M1, f) M2 and g) M4.

Figure 6 shows the stripping of the soluble TNF receptor I (sTNFRI) by immobilized TNF muteins, where a) relative sTNFRI, b) immobilized TNF mutein, c) null, d) M1, e) M2, f) M4, g) charge and h) eluted. Muteins 1, 2 and 4 were immobilized on Sepharose ™ 4B and normal human plasma reinforced with recombinant human sTNFRI was passed through the columns of the immobilized muteins. The stripping of sTNFRI from the plasma was measured by an enzyme-linked immunosorbent assay (ELISA). DETAILED DESCRIPTION OF THE INVENTION The present invention can be more easily understood with reference to the following detailed description of the preferred aspects of the invention and the Examples included therein and to the Figures and their previous and following description.

Before the present compounds, compositions, articles, devices and / or methods are disclosed and described, it should be understood that this invention is not limited to specific synthetic methods, specific nucleic acid molecules or wavelengths. particular laser, since they can, of course, vary. It should also be understood that the terminology used in this document is for the purpose of describing particular aspects only and is not intended to be limiting.

As used in the specification and the appended claims, the singular forms "a", "one" and "the" include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to a rbonucleic acid includes mixtures of rbonucleic acid molecules, reference to a probe includes mixtures of two or more said probes and the like.

Ranges can be expressed herein as from "about" a particular value and / or up to "about" another particular value. When said range is expressed, another aspect includes a particular value and / or the other particular value. Similarly, when the values are expressed as approximations, by the use of the "approximately" antecedent, it will be understood that the particular value forms another aspect. Furthermore, it will be understood that the endpoints of each of the ranges are significant both in relation to the other endpoint and independently of the other endpoint.

In this specification and in the claims that follow, reference will be made to a number of terms which will be defined to have the following meanings: "Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur and that the description includes cases where said event or circumstance occurs and cases where it does not. For example, the phrase "the sample may optionally contain more than one TNFa mutein" means that the sample may or may not contain more than one TNFa mutein and that the description includes both a sample containing a TNFα mutein and a sample containing more of a TNFa mutein Methods for reducing the levels of immune system inhibitors such as soluble TNF receptors in the circulation of a host mammal are provided, thereby enhancing an immune response capable of resolving a pathological condition or decreasing the severity of a pathological condition. By improving the magnitude of the host immune response, the methods disclosed avoid the problems associated with the repeated administration of chemotherapeutic agents which often have undesirable side effects, for example, chemotherapeutic agents used in cancer treatment.

The methods disclosed are generally achieved by: (a) obtaining a biological fluid from a mammal having a pathological condition, (b) contacting the biological fluid with a binding partner of mutein TNFa capable of selectively binding to an inhibitor target of the system immune such as a soluble TNF receptor to produce an altered biological fluid having a reduced amount of the target inhibitor of the immune system and thereafter (c) administering the altered biological fluid to the mammal.

As used herein, the term "immune system stimulator" refers to soluble mediators that increase the magnitude of an immune response, or that promote the development of particular immune mechanisms that are more effective in resolving a specific pathological condition. Examples of immune system stimulators include, but are not limited to, the pro-inflammatory mediators of factors a and b of tumor necrosis, interleukin-1, interleukin-2, interleukin-4, interleukin-5, interleukin-6, interleukin -8, interleukin-12, interferon-y, interferon-7 and the RANTES chemokines, macrophage inflammatory proteins 1-a and 1-β and the macrophage and activating factor, as discussed above.

As used herein, the term "immune system inhibitor" refers to a soluble mediator that decreases the magnitude of an immune response, or that discourages the development of particular immune mechanisms that are more effective in resolving a specific pathological condition or that encourage the development of particular immune mechanisms that are less effective in resolving a specific pathological condition. Examples of inhibitors of the host-derived immune system include interleukin-1 receptor antagonist, transforming growth factor-β, interleukin-4, interleukin-10 or the soluble receptors for interleukin-1, interleukin-2, interleukin-4, interleukin -6, interleukin-7, interferon-y and factors a and ß of tumor necrosis. In a particular aspect of the compositions, conjugates and methods disclosed, the inhibitor of the immune system may be a Type I (sTNFRI) or Type II soluble TNF receptor (sTNFRIl). Immune system inhibitors produced by microorganisms are also potential targets including , for example, soluble receptors for tumor necrosis factors a and B. As used herein, the term "target" inhibitor of the immune system refers to that inhibitor, or set of inhibitors, that is removed from the biological fluid by the methods disclosed, for example, sTNFRI and / or sTNFRIl.

As used herein, the term "soluble TNF receptor" refers to a soluble form of a receptor for TNFα and ββ. Two forms of the TNF receptor have been identified, the type I receptor (TNFRI), also known as TNF-R55 and the type II receptor (TNFRII), also known as TNF-R75, which are membrane proteins that bind to TNFa and TNF and mediate intracellular signaling. Both receptors also occur in a soluble form. The soluble form of the TNF receptor functions as an inhibitor of the immune system, as discussed above. As used herein, a soluble TNF receptor includes at least one of the soluble forms of TNFRI and TNFRII or any other type of TNF receptor. It is understood that, in the methods disclosed, the methods can be used to remove one or both types of TNF receptor depending on whether the mutein TNFa, or the plurality of muteins, used in the method binds to one or both types of receptors.

As used herein, the term "mammal" can be a human or a non-human animal, such as a dog, cat, horse, cattle, pig, sheep, non-human primate, mouse, rat, rabbit, or other mammal, for example. The term "patient" is used as a synonym of the term "mammal" to describe the compositions, conjugates and methods disclosed.

As used herein, the term "pathological condition" refers to any condition where the persistence within a host of an agent, immunologically distinct from the host, is a component or contributes to a disease state. Examples of such pathological conditions include, but are not limited to, those resulting from persistent viral, bacterial, parasitic, fungal and cancer infections. Among individuals exhibiting such chronic diseases, those in which the levels of immune system inhibitors are high are particularly appropriate for the disclosed treatment. The plasma levels of the immune system inhibitors can be determined using methods well known in the art (see, for example, Adolf and Apfler, supra, 1991). Those skilled in the art can easily determine pathological conditions that would benefit from stripping of immune system inhibitors in accordance with the present methods.

As used herein, the term "biological fluid" refers to a body fluid obtained from a mammal, e.g., blood, including whole blood, plasma, serum, lymphatic fluid or other types of bodily fluids. If desired, the biological fluid can be processed or fractionated, for example, to obtain an acellular component. As it relates to the compositions, conjugates and methods disclosed, the term "acellular biological fluid" refers to the acellular component of the circulatory system including plasma, serum, lymphatic fluid, or fractions thereof. The biological fluids can be removed from the mammal by any means or method known to those skilled in the art, including, for example, conventional apheresis methods (see, Apheresis: Principles and Practice, McLeod, Price and Drew, eds., AABB). Press, Bethesda, MD (1997)). The amount of biological fluid that is to be extracted from a mammal at a given time will depend on a number of factors, including the age and weight of the host mammal and the volume required to achieve a therapeutic benefit. As an initial guide, a volume of plasma (approximately 3-5 liters in an adult human) can be removed and, subsequently, stripped of the target inhibitor of the immune system in accordance with the present methods.

As used herein, the term "selectively binds" means that a molecule binds to a type of target molecule, but not substantially to other types of molecules. The term "specifically binds" is used interchangeably herein with "selectively binds." As used herein, the term "binding partner" is intended to include any molecule chosen for its ability to selectively bind to the target inhibitor of the immune system. The binding partner can be one that is naturally linked to the target inhibitor of the immune system. For example, factors a and ß of tumor necrosis can be used as a binding partner for sTNFRI. Alternatively, other binding partners, chosen for their ability to selectively bind to the target inhibitor of the immune system, may be used. These include fragments of the natural binding partner, monoclonal or polyclonal antibody preparations or fragments thereof or synthetic peptides. In a further aspect, the binding partner can be a TNFα mutein which can be a trimer, dimer or monomer.

As used herein, the term "mutein TNFa" refers to a variant of TNFα having one or more amino acid substitutions relative to a parent sequence and which retain binding activity specific to a TNF receptor, either soluble and / or membrane TNF. Generally, the muteins of the present invention have a single amino acid substitution relative to a parent sequence. Exemplary TNFa muteins include the human TNFa muteins termed muteins 1, 2, 3, 4, 5 and 6 (see Figure 3B), which are derived from human TNFa but have a single amino acid substitution relative to the wild-type sequence, such as it is discussed previously. It is understood that analogous muteins of species other than human include similarly, for example, mutein analogs for muteins 1, 2, 3, 4, 5 and 6 in the other mammalian species shown in Figure 3A or other mammalian species. These and other muteins, as described in more detail below, are included within the meaning of the TNFα mutein of the invention. In addition, TNFα muteins can include mutant or altered forms of TNFα that, for example, have reduced ability to form multimers or are coupled to form a dimer. The reported alterations of native TNFα can be used separately or together in any combination. In addition, alterations in muteins 1, 2, 3, 4, 5 and 6 can be used together or separately and each or all can be used together with alterations that result in monomer or dimer formation. For example, an altered monomer of wild-type TNFα can be coupled with a second altered monomer of wild-type TNFα to form a dimer. In a further aspect, an altered monomer of wild type TNFa can be coupled with an amino acid sequence, for example mutein 1, to form a dimer. Thus, "TNFa muteins" can include an altered monomer of TNFa, a monomer comprising a single mutation in wild-type TNFα, for example mutein 1, mutein 2, mutein 3, mutein 4, mutein 5, mutein 6 or any other mutated amino acid sequence described herein, a dimer comprising two identical amino acid sequences and a dimer containing two non-identical amino acid sequences. For example, a TNFα mutein may include a dimer comprising two identical altered monomers of wild-type TNFα or two non-identical altered monomers of wild-type TNFα. In a further aspect, a TNFα mutein may include a dimer comprising two amino acid sequences that contain an identical amino acid mutation. In a further aspect, a TNFα mutein may include a dimer comprising two amino acid sequences having non-identical amino acid mutations. In addition, a TNFα mutein may include a dimer comprising an amino acid sequence of an altered form of wild-type TNFα and an amino acid sequence containing a single amino acid mutation, for example an amino acid sequence identified by SEQ ID NO. : 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6. SEQ ID NO: 7, SEQ ID NO: 8 or any mutated amino acid sequence disclosed herein.

A TNFa mutein may be monomeric, dimeric or trimeric. That is, a TNFa mutein may have a monomeric form, a dimeric form or a trimeric form. Native TNFa exists as a homotrimer of three kDa subunits, as do the TNFa muteins exemplified herein. Other TNFα muteins produced using the methods described herein may also exist as trimers. Linking TNFα or TNFα muteins to monovalent soluble TNFR involves only two of the three monomers present in TNFα or the mutein TNFα trimer. While TNFa or the mutein TNFa trimer can bind to three soluble monovalent TNFR molecules, the binding to each is an independent event which is governed solely by affinity and which does not allow a contribution for avidity. Therefore, the binding strength by a dimeric form of TNFα or mutein TNFα to soluble TNFR does not decrease in relation to the trimeric form of TNFα or TNFα muteins.

Compositions and methods are provided for stimulating or improving an immune response in a mammal. The invention advantageously uses ligands that bind to inhibitors of the immune system to counteract or decrease the termination effect of immune system inhibitors on the immune response. Said ligands, also referred to herein as "binding partners", can be attached to a solid support to allow removal of an inhibitor of the immune system from a biological fluid.

A binding partner particularly useful in the present invention is a ligand that binds with high affinity to an inhibitor of the immune system, for example, soluble TNF receptor and in particular sTNFRI and / or sTNFRII. Another useful feature of a binding partner is the lack of direct toxicity. For example, a binding partner that lacks or has reduced TNF agonist activity is particularly useful. Generally, even when a ligand such as a binding partner is covalently bound to a solid support, a certain percentage of bound ligand will leach from the support, for example, via chemical reactions that break the covalent bond or protease activity present in a biological fluid. In that case, the ligand will leach in the biological fluid that is being processed and so will be returned to the patient. Therefore, it is advantageous to use a ligand that has affinity with an inhibitor of the immune system but has decreased ability to stimulate a biological response, that is, it has decreased or low agonist activity. In this case, even if some of the ligand leaches into the processed biological fluid, the ligand will still exhibit low biological activity with respect to the signaling of the membrane receptor when it is introduced into the patient.

Yet another useful feature of a binding partner is the lack of indirect toxicity, for example, immunogenicity. As discussed above, it is common for a bound ligand to leach from a matrix, which results in the ligand being present in the processed biological fluid. When the biological fluid returns to the patient, this results in the introduction of a low level of the ligand to the patient. If the ligand is immunogenic, an immune response against the ligand can be stimulated, resulting in undesirable immune responses, particularly in a patient in whom the process is being repeated. Therefore, a ligand having low immunogenicity would minimize any undesirable immune response against the ligand. As disclosed herein, a ligand particularly useful as a binding partner of the invention is derived from the same species of the patient being treated. For example, to treat a human, a human TNFα mutein can be used as the binding partner, which is expected to have low immunogenicity given the homology of endogenous TNFα. Similarly, muteins derived from another mammalian species can be used in the respective species.

As disclosed herein, TNFα muteins are binding partners particularly useful in methods of the invention. A number of TNFα muteins have previously been described (see, for example, Van Ostade et al., Protein Enq. 7: 5-22 (1994), Van Ostade et al., EMBO J. 10: 827-836 (1991)). , Zhang et al., J. Biol. Chem. 267: 24069-24075 (1992), Yamagishi et al., Protein Enq. 3: 713-719 (1990), each of which is incorporated herein by reference. reference). Specific exemplary muteins include the human TNFα muteins shown in Figure 3B.

There are several advantages to using TNFα muteins as binding partners in the present invention. Although TNFa muteins may exhibit lower binding activity for TNF receptors, some TNFa muteins bind only 5 to 17 times less effectively than native TNFa. Said binding affinity, however reduced in relation to the native TNFa, can still be an effective binding partner in the present invention (see Example 3). Another advantage of using TNFα muteins is that some exhibit decreased signaling through membrane receptors, eg, decreased cytotoxic activity or in vivo toxicity, relative to native TNFα. In particular, muteins 1, 2, 3, 4, 5 and 6 exhibit a 200 to 10,000 fold decrease in cytotoxicity (see below and Van Ostade, supra, 1994; Yamagishi et al., Supra, 1990; Zhang et al. ., supra, 1992). Thus, even when the binding affinity is reduced to 10 to 17 times, there may be a 200 to 10,000 fold decrease in signaling through the membrane receptors, eg, decreased cytotoxic activity or in vivo toxicity. As discussed above, such reduced signaling through membrane receptors, eg, reduced cytotoxicity or in vivo toxicity, is advantageous in view of the possible leaching of the ligand from a matrix and introduction of low levels in a patient when a fluid altered biological returns to the patient. In addition, the dimeric fusion proteins of TNFα or dimeric forms of mutein TNFα can be used in the methods disclosed. Such dimeric fusions are useful because they (1) bind to the soluble TNFR receptor with sufficient affinities to remove soluble TNFR from a biological fluid and (2) because they exhibit reduced binding to, or signaling through, membrane TNFR. in relation to wild-type TNFa, thus reducing or eliminating toxicity.

An additional advantage of using TNFa muteins is that they have a native structure. Because the muteins are highly homologous to the native TNFa sequence, these muteins can be bent into a native structure that retains TNF receptor binding activity. Said native structure means that the same amino acid residues are exposed on the surface of the molecule as in native TNFα, except possibly for the amino acid residue muante Said native doubling means that the TNFa muteins should have little or no immunogenicity in the respective mammalian species.

As discussed herein, particularly useful muteins are the human muteins 1, 2, 3, 4, 5 and 6 (Figure 3B) and the analogous muteins in other mammalian species. Mutein 1 is a single amino acid substitution relative to wild-type human TNF [alpha] of Arg31 with Pro (Zhang et al., Supra, 1992). This mutein exhibits approximately 10-fold lower binding activity than membrane TNFR and approximately 10,000-fold lower cytotoxicity relative to native TNFα. Mutein 2 is a single amino acid substitution relative to wild-type human TNF [alpha] of Asn34 with Tyr (Yamagishi et al., Supra, 1990; Asn32 in the numbering system of Yamagishi et al.). This mutein exhibits approximately 5-fold lower binding activity to membrane TNFR and approximately 12,500-fold lower cytotoxicity relative to native TNFα. Mutein 3 is a single amino acid substitution relative to wild-type human TNFα of Pro1 17 with Leu (Yamagishi et al., Supra, 1990; Pro115 in the numbering system of Yamagishi et al.). This mutein exhibits approximately 12 times lower binding activity to the membrane TNFR and approximately 1400 times lower cytotoxicity. Mutein 4 is a single amino acid substitution relative to wild-type human TNFα of Ser147 with Tyr (Zhang et al., Supra, 1992). This mutein exhibits approximately 14-fold lower binding activity to membrane TNFR and approximately 10,000-fold lower cytotoxicity relative to native TNFα. Mutein 5 is a single amino acid substitution relative to wild-type human TNFα of Ser95 with Tyr (Zhang et al., Supra, 1992). This mutein exhibits approximately 17-fold lower binding activity to membrane TNFR and approximately 200-fold lower cytotoxicity relative to native TNFα. Mutein 6 is a single amino acid substitution relative to wild-type human TNF [alpha] of Tyr115 with Phe (Zhang et al., Supra, 1992). This mutein exhibits approximately 17-fold lower binding activity to membrane TNFR and approximately 3,300-fold lower cytotoxicity relative to native TNFα. As disclosed herein, it is understood that analogous muteins can be generated in other species of mammals by making the same substitutions of amino acids in the analogous position of the respective species.

Although muteins 1, 2 and 4, as well as other TNFa muteins, were previously known and characterized with respect to multivalent membrane receptor binding, it was previously unknown whether these TNFa muteins would bind to monovalent soluble TNF receptors. As disclosed herein, the TNFα muteins bind with sufficient affinity to strip the soluble TNF receptor from the plasma (see Examples 3 and 6). These results indicate that the TNFa muteins can be an effective binding partner for stripping the soluble TNF receptor from a biological fluid.

It is understood that additional TNFa muteins to the specific muteins exemplified herein can be used in methods of the invention. TNFa from various mammalian species show a high degree of amino acid identity (see Figures 3A and 3B, conserved sequence SEQ ID NO: 1, Van Ostade et al., Supra, 1994). As described by Van Ostade et al. (supra, 1994), a conserved amino acid sequence TNFa was identified through 11 mammalian species. Conserved amino acid residues are conserved across all 11 species shown or have only a single species showing variation in that position (see Figure 3A and Van Ostade et al., Supra, 1994). Thus, a mutein comprising the conserved sequence mentioned as SEQ ID NO: 1 is provided.

A person skilled in the art can easily determine additional muteins suitable for use in the compositions, conjugates and methods disclosed. As discussed above, TNFα muteins that have relatively high affinity to TNF receptors and decreased signaling through membrane receptors, eg, decreased cytotoxicity or live toxicity, relative to native TNFα, are particularly useful in compositions, conjugates and disclosed methods. A person skilled in the art can easily determine additional appropriate TNFa muteins based on well-known methods by those with skill in the art. Methods for introducing amino acid substitutions in a sequence are well known to those skilled in the art (Ausubel et al., Current Protocols in Molecular Bioloqy (Supplement 50), John Wiley &Sons, New York (2001), Sambrook and Russell, Molecular Clonine: A Laboratorv Manual, 3rd ed., Cold Spring Harbor Press, Cold Spring Harbor (2001), US Patent Nos. 5,264,563 and 5,523,388). The generation of mutein TNFa has been previously described (Van Ostade et al., Supra, 1994, Van Ostade et al., Supra, 1991, Zhang et al., Supra, 1992, Yamagishi et al., Supra, 1990). In addition, a person skilled in the art can easily determine the binding and cytotoxicity and / or in vivo toxicity of candidate muteins to ensure suitability for use in the disclosed method (Van Ostade et al., Supra, 1994, Van Ostade et al. al., supra, 1991; Zhang et al., supra, 1992; Yamagishi et al., supra, 1990).

TNFa muteins of particular interest for use in the disclosed compositions, conjugates and methods, in addition to having relatively high affinity for TNF receptors and reduced signaling through membrane receptors, eg, reduced cytotoxicity or in vivo toxicity, are those that have amino acid substitutions in three TNFa regions; region 1, amino acids 29-36; region 2, amino acids 84-91 and region 3, amino acids 143-149 (numbering as shown in Figure 3A). Muteins 1, 2 and 4 are examples of muteins that have simple amino acid substitutions in these regions. Region 1 corresponds to amino acids 29-36, LNRRANAL residues (amino acids 29-36 of SEQ ID NO: 2) of human TNFa. Region 2 corresponds to amino acids 84-91, residues AVSYQTKV (amino acids 84-91 of SEQ ID NO: 2) of human TNFa. Region 3 corresponds to amino acids 143-149, residues DFAESG (SEQ ID NO: 20) of human TNFa. In addition to the TNFα muteins disclosed herein, other TNFα muteins can be generated, for example, by introducing single amino acid substitutions in regions 1, 2 or 3 and look for binding activity and cytotoxic activity and / or in vivo toxicity as described in this document (see also Van Ostade et al., supra, 1991; Zhang et al., Supra, 1992; Yamagishi et al., Supra, 1990). Methods for introducing amino acid substitutions in an amino acid residue or region particular are well known to those skilled in the art (see, for example, Van Ostade et al., supra, 1991; Zhang et al., supra, 1992; Yamagishi et al., supra, 1990; US Patents Nos. 5,264,563 and 5,523,388). For example, each of the other 19 amino acids relative to a native sequence can be introduced at each of the positions in regions 1, 2 and 3 and look for binding activity and / or signaling activity, eg, cytotoxic activity or toxicity in live, for soluble receptor and / or membrane bound TNF. This would only require the generation of approximately 420 mutants (19 substitutions of single amino acids in each of the 22 positions in regions 1, 2 and 3), a number that can be easily generated and searched by well-known methods. Those having the desired characteristics as disclosed herein, for example, specific binding activity for soluble TNF receptor and reduced signaling through the membrane TNF receptor, can be selected as a useful TNFα mutein in the compositions, conjugates and methods disclosed.

A TNFα mutein having the consensus sequence of the SEQ is also provided ID NO: 9 (Figure 3C). In one aspect, a TNFa mutein comprises the consensus sequence SEQ ID NO: 9, wherein Xi is an amino acid selected from Leu and Val, wherein X2 is a 2 or 3 amino acid peptide having Gln or Arg in the 1-position. , Asn, Ala or Thr in position 2 and Ser, Leu, Pro or absence in position 3, for example, selected from GlnAsnSer, ArgAlaLeu, ArgThrPro, GlnAlaSer and GlnThr, where X3 is an amino acid selected from Asp and Asn, wherein X4 is a 5 amino acid peptide having His, Pro, Leu, He or Val in position 1, Gln, Glu, Ser, Asn or Lys in position 2, Val, Ala or Ser in position 3, Glu or Pro at position 4 and Glu or Gly at position 5, for example, selected from HisGlnVaIGluGlu (SEQ ID NO: 21), HisGlnAlaGluGlu (SEQ ID NO: 22), ProGInVaIGluGly (SEQ ID NO: 23), ProGluAlaGluGly (SEQ ID NO: 24), LeuSerAlaProGly (SEQ ID NO: 25), HeSerAlaProGly (SEQ ID NO: 26), ProGInAlaGluGly (SEQ ID NO: 27), UeAsnSerProGly (SEQ ID NO: 28) and ValLysAlaGluGly (SEQ ID NO : 29), wherein X5 is an amino acid selected from Glu, Gln and Arg, wherein Xe is a 4 amino acid peptide having Leu, Gly, Trp or Gln at position 1,, Ser, Asp or Asn at the position 2, Gln, Arg, Ser or Gly in position 3 and Arg or Tyr in position 4, for example, selected from LeuSerGInArg (SEQ ID NO: 30), LeuSerArgArg (SEQ ID NO: 31), GlyAspSerTyr (SEQ ID NO: 32), LeuSerGIyArg (SEQ ID NO: 33). TrpAspSerTyr (SEQ ID NO: 34), GInSerGIyTyr (SEQ ID NO: 35) and LeuAsnArgArg (SEQ ID NO: 36), wherein X7 is an amino acid selected from Leu, Met and Lys, wherein X8 is a peptide of two amino acids having Met or Val in position 1 and Asp, Lys, Glu or Gln in position 2, for example, selected from MetAsp, MetLys, ValGlu, ValLys and ValGIn, where X9 is an amino acid selected from Lys, Thr, Glu and Arg, wherein X10 is an amino acid selected from Val, Lys and He, where X is a 2 amino acid peptide having Ala, Ser, Thr or Leu in position 1 and Asp or Glu in position 2, for example , selected from AlaAsp, SerAsp, ThrAsp, LeuASp, AlaGlu and SerGIu, wherein X12 is an amino acid selected from Lys, Ser, Thr and Arg, wherein X13 is an amino acid selected from Gln and His, wherein X1 is a peptide of 4 or 5 amino acids that have Asp, Ser or Pro in position 1, Val, Tyr, Pro or Thr in position 2, Val, Pro, His or Asn in position 3, Leu or Val in the position n 4 and Leu, Phe or absence in position 5. for example, selected from AspValValLeu (SEQ ID NO: 37), AspTyrValLeu (SEQ ID NO: 38), SerTyrValLeu (SEQ ID NO: 39), ProProProVal (SEQ ID NO : 40), SerThrHisValLeu (SEQ ID NO: 41), SerThrProLeuPhe (SEQ ID NO: 42) and SerThrAsnValPhe (SEQ ID NO: 43), where X15 is an amino acid selected from Val e He, where X 6 is an amino acid selected from Phe, He and Leu, where X17 is an amino acid selected from He and Val, where X1B is a 2 amino acid peptide having Gln or Pro in position 1 and Glu, Asn, Thr or Ser in position 2, for example , selected from GlnGlu, ProAsn, GlnThr and ProSer, wherein X19 is an amino acid selected from Leu and He, wherein X2o is a 3 amino acid peptide having Pro, His or Gln at position 1, Lys, Arg or Thr in position 2 and Asp or Glu at position 3, for example, selected from ProLysAsp, HisArgGlu, GlnArgGlu and HisThrGIu, where X2i is an amino acid selected from Gly, Glu, Gln and Trp or absent, where X22 is an amino acid selected from Leu, Pro and Ala, wherein X23 is an amino acid selected from Leu and Gln, wherein X24 is an amino acid selected from Gly and Asp, wherein X25 is an amino acid selected from Gln, Leu and Arg, wherein X26 is an amino acid selected from Ala and Thr, where X27 is an amino acid selected from Val e He, where X28 is a n amino acid selected from Leu, Gln and Arg, wherein X29 is an amino acid selected from Lys, Glu, Ala, Asn and Asp, wherein X30 is an amino acid selected from Phe, Me, Leu and Tyr and wherein X31 is an amino acid selected from Val e lie (see Figure 3A, Van Ostade et al., Supra, 1994). Said mutein TNFa consensus is expected to exhibit binding activity to the TNF receptor and such activity can be readily determined by those skilled in the art using well known methods, as disclosed herein.

In addition to the variable positions described above, it is understood that a TNFα mutein may additionally include variable amino acids in the conserved sequence referred to as SEQ ID NO: 1. As shown in Figure 3A and as discussed above, the conserved TNFα sequence includes certain positions where one of the mammal species shown differs from the other ten. For example, the amino acid conserved at position 2, Arg, is Leu in the dog (Figure 3A). Thus, a TNFa mutein may include a substitution of Leu at position 2 with the rest of the conserved sequence mentioned as SEQ ID NO: 1. Similarly, substitutions of other "conserved" positions, where at least one of the species has a Amino acid substitution relative to the conserved sequence is included as mutein TNFa. For example, a mutein TNFα can have the corresponding substitution of mutein 1, that is, Arg31 Pro and the substitution in the sequence conserved in the variable positions, as described above represented by X and / or the substitution in a conserved position that varies in a single species. In addition, a TNFα mutein may include conservative amino acid substitutions relative to the conserved sequence or sequence of a particular species of TNFα. Such TNFα muteins can be readily recognized by a person skilled in the art based on the desired characteristics of a TNFα mutein, as disclosed herein.

Any of the TNFα muteins disclosed herein may be modified to include an N-terminal deletion. As discussed in Van Ostade. { supra, 1994), short eliminations in the N-terminals of TNFa retained activity, while that the elimination of 17 amino acids in the N-terminal resulted in a loss of activity. Therefore, it is understood that the disclosed TNFα muteins also include TNFα muteins that have N-terminal deletions that retain activity. Said TNFa muteins may include, for example, an N-terminal deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids. In addition, a person skilled in the art can easily determine if additional N-terminal deletions can be incorporated into a TNFα mutein by making the elimination mutations and look for the desired characteristics, as disclosed herein.

A variety of TNFα muteins is provided as disclosed herein. Generally, a particularly useful TNFα mutein is about 2 times, about 3 times, about 4 times, about 5 times, about 6 times, about 7 times, about 8 times, about 9 times, about 10 times, about 11 times, about 12 times. times, about 13 times, about 14 times, about 15 times, about 16 times, about 17 times, about 18 times, about 19 times, about 20 times, about 25 times, about 30 times or even more times reduced binding affinity for receptors TNF, particularly membrane-bound TNF receptors, in relation to native / wild-type TNFa. Said reduced binding affinity may be, but is not necessarily, exhibited towards sTNFR. Also, a particularly useful TNFα mutein has approximately 5 times, approximately 10 times, approximately 50 times, approximately 100 times, approximately 150 times, approximately 200 times, approximately 300 times, approximately 500 times, approximately 1000 times, approximately 2000 times, approximately 3000 times, approximately 4000 times, approximately 5000 times, approximately 6000 times, approximately 7,000 times, approximately 8,000 times, approximately 9,000 times, approximately 10,000 times, approximately 20,000 times, approximately 30,000 times, approximately 50,000 times or even more times reduced signaling through the membrane receptors, for example, reduced cytoxicity or toxicity in vivo, in relation to native / wild-type TNFa. It is understood that a TNFα mutein may have reduced binding affinity and / or reduced cytoxicity, as discussed above and disclosed herein.

A conjugate comprising a tumor necrosis factor a mutein (TNFa) fixed to a substrate is provided herein. In a further aspect, the mutein TNFα of the conjugate comprises the conserved sequence mentioned as SEQ ID NO: 1.

A conjugate is further provided wherein the mutein TNFa has the consensus sequence SEQ ID NO: 9, wherein Xi is an amino acid selected from Leu and Val, wherein X2 is a peptide of 2 or 3 amino acids having Gln or Arg in the position 1, Asn, Ala or Thr in position 2 and Ser, Leu, Pro or absence in position 3, for example, selected from GlnAsnSer, ArgAlaLeu, ArgThrPro, GInAlaSer and GInThr, where X3 is an amino acid selected from Asp and Asn, where X is a 5 amino acid peptide having His, Pro, Leu, He or Val in position 1, Gln, Glu, Ser, Asn or Lys in position 2, Val, Ala or Ser in position 3 , Glu or Pro at position 4 and Glu or Gly at position 5, for example, selected from HisGlnVaIGluGlu (SEQ ID NO: 21), HisGlnAlaGluGlu (SEQ ID NO: 22), ProGInVaIGluGly (SEQ ID NO: 23), ProGluAlaGluGly (SEQ ID NO: 24), LeuSerAlaProGly (SEQ ID NO: 25), HeSerAlaProGly (SEQ ID NO: 26), ProGInAlaGluGly (SEQ ID NO: 27), HeAsnSerProGly (SEQ ID NO: 28) and ValLy sAlaGluGly (SEQ ID NO: 29), wherein X5 is an amino acid selected from Glu, Gln and Arg, wherein Xe is a 4 amino acid peptide having Leu, Gly, Trp or Gln at position 1, Ser, Asp or Asn in position 2, Gln, Arg, Ser or Gly in position 3 and Arg or Tyr in position 4, for example, selected from LeuSerGInArg (SEQ ID NO: 30), LeuSerArgArg (SEQ ID NO: 31), GlyAspSerTyr (SEQ ID NO: 32), LeuSerGIyArg (SEQ ID NO: 33), TrpAspSerTyr (SEQ ID NO: 34), GlnSerGIyTyr (SEQ ID NO: 35) and LeuAsnArgArg (SEQ ID NO: 36), wherein X7 is an amino acid selected from Leu, Met and Lys, wherein Xa is a two amino acid peptide having Met or Val in position 1 and Asp, Lys, Glu or Gln in position 2, for example, selected from MetAsp, MetLys, ValGlu, ValLys and ValGIn, where Xg is an amino acid selected from Lys, Thr, Glu and Arg, where X10 is an amino acid selected from Val, Lys e lie, wherein X is a 2 amino acid peptide having Ala, Ser, Thr or Leu in position 1 and Asp or Glu in position 2, for example, selected from AlaAsp, SerAsp, Thr Asp , LeuASp, AlaGlu and SerGIu, wherein X12 is an amino acid selected from Lys, Ser, Thr and Arg, wherein Xi3 is an amino acid selected from GIn and His, wherein X14 is a 4 or 5 amino acid peptide having Asp, Ser or Pro in position 1, Val, Tyr, Pro or Thr in position 2, Val, Pro, His or Asn in position 3, Leu or Val in position 4 and Leu, Phe or absence in position 5, for example, selected from AspValValLeu (SEQ ID NO: 37), AspTyrValLeu (SEQ ID NO: 38), SerTyrValLeu (SEQ ID NO: 39), ProProProVal (SEQ ID NO: 40), SerThrHisValLeu (SEQ ID NO: 41), SerThrProLeuPhe (SEQ ID NO: 42) and SerThrAsnValPhe (SEQ ID NO: 43), where X 5 is an amino acid selected from Val e He, where X16 is an amino acid selected from Phe, He and Leu, where X17 is a amino acid or selected from He and Val, wherein X18 is a 2 amino acid peptide having GIn or Pro at position 1 and Glu, Asn, Thr or Ser at position 2, for example, selected from GlnGlu, ProAsn, GInThr and ProSer , wherein XiS is an amino acid selected from Leu e He, where X2o is a 3 amino acid peptide having Pro, His or GIn in position 1, Lys, Arg or Thr in position 2 and Asp or Glu in position 3, for example, selected from ProLysAsp, HisArgGlu, GlnArgGlu and HisThrGIu, wherein X2i is an amino acid selected from Gly, Glu, GIn and Trp or is absent, wherein X22 is an amino acid selected from Leu, Pro and Ala, wherein X23 is an amino acid selected from Leu and GIn, wherein X24 is an amino acid selected from Gly and Asp, wherein X25 is an amino acid selected from GIn, Léu and Arg, wherein X26 is an amino acid selected from Ala and Thr, wherein X27 is an amino acid selected from Val e He , wherein X2e is an amino acid selected from Leu, GIn and Arg, wherein X29 is an amino acid selected from Lys, Glu, Ala, Asn and Asp, wherein X30 is an amino acid selected from Phe, He, Leu and Tyr and in where X31 is an amino acid selected from Val e He.

In a further aspect, a conjugate is provided wherein the mutein TNFa has an amino acid substitution in a region of TNFa selected from region 1 of amino acids 29-36, region 2 of amino acids 84-91 and region 3 of amino acids 143 -149 of human TNFα (SEQ ID NO: 2) of the analogous position of TNFα from another species.

Also, a conjugate is provided wherein the mutein TNFa is selected from mutein 1 (SEQ ID NO: 3), mutein 2 (SEQ ID NO: 4), mutein 3 (SEQ ID NO: 5), mutein 4 (SEQ ID NO. : 6), mutein 5 (SEQ ID NO: 7) and mutein 6 (SEQ ID NO: 8). In a particular aspect, a conjugate is provided wherein the mutein TNFa is selected from mutein 1 (SEQ ID NO: 3), mutein 2 (SEQ ID NO: 4) and mutein 4 (SEQ ID NO: 6). In the dimeric form, a TNFα mutein may comprise two identical amino acid sequences or two non-identical amino acid sequences. The mutein TNFa of the conjugate can be derived from a selected species, for example from human, dog, cat, horse, sheep, goat, pig, cow, rabbit and rat.

A method for stimulating an immune response in a mammal having a pathological condition is further provided. The method can include the steps of obtaining a biological fluid from a mammal, contacting the biological fluid with a tumor necrosis factor a (TNFa) mutein that has specific binding activity for a soluble tumor necrosis factor receptor ( TNFR), removing the TNFα mutein bound to soluble TNFR from the biological fluid to produce an altered biological fluid having a reduced amount of soluble TNFR and administering the altered biological fluid to the mammal. The biological fluid may be, for example, blood, plasma, serum or lymphatic fluid, including whole blood. In a further aspect, a method using whole blood as the biological fluid may further include the step of separating the whole blood into a cellular component and an acellular component or a fraction of the acellular component, wherein the acellular component or the fraction of the component acellular contains a soluble TNFR. The method may further include the step of combining the additional component with the altered acellular component or altered fraction of the acellular component to produce altered whole blood, which may be administered to the mammal as the altered biological fluid. Accordingly, the cellular component and the altered acellular component or the altered fraction of the acellular component can be administered separately to the mammal.

A TNFa mutein may have specific binding activity for a simple type of Soluble TNFR, for example sTNFRI or sTNFRII. Alternatively, the mutein TNFa may have specific binding activity for more than one type of soluble TNFR, for example, both sTNFRI and sTNFRII.

Various mixtures of bonding partners can be used. For example, a mixture may be composed of multiple binding partners that selectively bind to an objective inhibitor of the immune system. Another mixture can be composed of multiple binding partners, each of which selectively binds to different target inhibitors of the immune system. Alternatively, the mixture can be composed of multiple binding partners that selectively bind to different target inhibitors of the immune system. For example, the mixture may contain more than one mutein TNFa. In addition, the multiple TNFa muteins can bind specifically to a single type of soluble TNF receptor or can bind to more than one type of TNF receptor, for example, sTNFRI and sTNFRII.

In a further aspect, the biological fluid can be contacted with a plurality of TNFa muteins. Therefore, the plurality of muteins TNFa may have specific binding activity for a single type of soluble TNFR, for example, sTNFRI or sTNFRII. Alternatively, the plurality of muteins TNFa may have specific binding activity for more than one type of soluble TNFR, ie, sTNFRI and sTNFRII.

When it is desirable to increase the molecular weight of the binding partner / inhibitor complex of the immune system, the binding partner can be conjugated to a carrier. Examples of such carriers include, but are not limited to, proteins, complex carbohydrates, and synthetic polymers such as polyethylene glycol.

As used herein, "functionally active binding sites" of a binding partner refers to sites that are capable of binding to one or more target inhibitors of the immune system.

One method to generate a dimeric form of a TNFα or a TNFα mutein is to produce a fusion protein that covalently binds a TNFα or a mutein TNFα monomer to an antibody heavy chain constant region. A particularly useful method for generating a dimeric TNFα is to fuse a TNFα with each of the two heavy chain constant regions involved in the formation of the Fe dimeric portion of an antibody. As these heavy chain constant region monomers come together to form a stable Fe structure, they will cause the associated TNFa or mutein TNFa molecules to also dimerize. In one aspect, the two amino acid sequences of the dimeric form are identical; In another aspect, the two amino acid sequences of dimeric form are non-identical. In yet another aspect, a fusion protein can be produced in which the amino terminus of the mutein TNFa is fused with the carboxy-terminus of an intact heavy chain constant region. TNFa has been fused at its amino terminus into a variety of fusion proteins without significant loss of biological activity. Said heavy chain mutein TNFa fusion protein can be coupled with light chains of intact antibody to form a molecule that would neutralize soluble TNFR and possess limited immunogenicity in a mammal from which the antibody and TNFa sequences are derived. In another aspect, the heavy chain constant region can be truncated amino terminus of the cartilage region, thus providing two sites to which a TNFa or mutein monomer TNFa can be fused at its carboxy terminus. Fusion proteins that involve the carboxy terminals of TNFα have been produced, but typically these have resulted in significant losses in the biological activity of the TNFα component of the fusion protein. The losses observed in the activity result from the fact that the carboxyl group of the carboxyl terminal amino acid of TNFa (Leu157) forms a pair with Lys11 in an adjacent monomer, thus stabilizing the formation of the trimer (Eck and Sprang, J. Biol, Chem. 264 (29): 17595-17605 (1989 ), which is incorporated herein by reference). If the formation of a peptide bond utilizing the carboxyl group of Leu157 leads to the decreased binding of the fusion protein to the soluble TNFR, one or more amino acids can be inserted at the link between the amino termini of the heavy chain constant region and the carboxy terminals of the mutein TNFa, the amino acids having R-groups containing carboxyl functionalities (e.g., Asp or Glu). In yet another aspect, other dimeric plasma proteins, including either homodimers and heterodimers, can be fused to the amine or carboxy terminus of a TNFα mutein as described above.

Alternatively, a TNFa monomer can be cross-linked with a plasma protein, for example, an antibody or serum albumin, using well-known chemical cross-linking methods. Such methods are well known as taught, for example, in Hermanson, Bioconiuaate Techniaues. Academic Press, San Diego (1996).

Also provided are the monomeric forms of TNFα and TNFα muteins, which can be generated to further reduce the binding to, and signaling through, the membrane TNFR. The monomeric form of TNFa is anticipated to have the lowest binding strength of any TNFa isoform for membrane TNFR due to the absence of any contribution to avidity. further, monomeric TNFa anticipates the signal through membrane TNFR significantly less well than trimeric TNFα since the ability of monomeric TNFα to cross-link membrane TNFR is reduced. As noted above, the binding of TNFα or mutein TNFα to monovalent soluble TNFR involves only two of the three monomers present in the TNFα or mutein monomer TNFα. Thus, TNFa and TNFa muteins must be multimerized to at least one dimeric state to bind to soluble TNFR. The multimerization of the monomeric TNFα or the mutein TNFα is easily achieved by its covalent conjugation to a solid support when creating the absorbent matrix that is disclosed in the present document. Thus, two adjacent molecules of monomeric TNFα or mutein TNFα, once immobilized in proximity to one another on the solid support, can bind to soluble TNFR. At the time of the dissociation of the solid support, however, these ligands will return to the monomeric state and, thus, will bind and signal poorly through the membrane TNFR.

One method for generating a monomeric form of a TNFα or a TNFα mutein is to produce a fusion protein in which TNFα or the mutein monomer TNFα is fused at its carboxy terminus with amino acids not present in wild type TNF. As noted above, terminal-carboxy fusions of TNFα have been produced, but typically these have resulted in significant losses in the biological activity of the TNFα component of the fusion protein. The observed losses in activity result from the fact that the carboxyl group of the carboxy terminal amino acid of TNFa (Leu157) forms a pair with Lys11 in an adjacent monomer, thus stabilizing the trimeric formation (Eck and Sprang, J. Biol .. Quím 264 (29): 17595-17605 (1989), which is incorporated herein by reference). The incorporation of additional amino acids, which extend the carboxy terminus of the molecule at a sufficient distance to prevent the pairing of ions with Lys11 in an adjacent monomer, should decrease the trimeric formation. Amino acids that can be fused to the carboxy terminus of TNFα or mutein TNFα may include a purification tag (eg, polyhistidin or GST) or any random amino acid sequence that allows for proper folding of the monomer and that is preferably not immunogenic .

In another aspect, the mutations can be introduced into TNFα or the mutein TNFα portion of a dimeric fusion protein, said mutations being designed to reduce the ability of the TNFα or dimerized TNFα mutein to associate with a wild-type TNFα monomer. The combination of TNFα or the dimeric TNFα mutein with a TNFα monomer would restore the trimeric structure and potentially increase the ability of the fusion protein to bind to the membrane TNFR and, thus, contribute to toxicity, if the protein fusion was to be released from the absorbent matrix and returned to the patient. The introduction of mutations in residues that normally form ion pairs that contribute to the assembly of TNFα or trimeric TNFα muteins (eg, Lys98 and Glu116 or Lys and Leu157, Eck and Sprang, supra, 1989) would reduce or eliminate the ability of such muteins to associate with the wild type TNFa monomers. As discussed above, the fusion of TNFα or TNFα muteins at its carboxy terminus Leu157 with an immunoglobulin heavy chain or other fusion partner can serve to prevent association with a wild-type TNFα monomer by preventing the formation of an ion pair with Lys11 in an adjacent sub-unit.

Methods for the production of various binding partners useful with the disclosed compositions, conjugates and methods are well known to those skilled in the art. Such methods include, for example, recombinant DNA and synthetic techniques, or a combination thereof. Binding couples such as TNFa muteins can be expressed in prokaryotic or eukaryotic cells, for example, from mammals, insects, bacteria and the like. If desired, the codons can be changed to reflect any codon tilt in a host species used for expression.

A binding partner, such as the TNFa mutein, can be fixed to an inert medium to form an absorbent matrix (Figure 1). The mutein TNFa may be, for example, covalently fixed to a substrate such as an inert medium. As used herein, the term "inert medium" is intended to include solid supports to which bonding partners can be attached. Particularly useful supports are materials that are used for such purposes, including, for example, hollow fibers with cellulose base, synthetic hollow fibers, silica-based particles, flat or pleated membranes, macroporous beads, agarose-based particles and the like. The inert medium can be in the form of an account, for example, a macroporous account or a non-porous account. Exemplary macroporous beads include, but are not limited to, naturally occurring materials such as agarose, cellulose, controlled pore glass or synthetic materials such as polyacrylamide, crosslinking agarose (such as Trisacryl ™, Sephacryl, Actigel ™ and Ultrogel ™), azlactone, polymethacrylate, polystyrene / divinylbenzene and the like. In one aspect, a macroporous account comprises Actigel ™. Exemplary non-porous beads include, but are not limited to, silica, polystyrene, latex and the like. The fibers and hollow membranes can also be composed of natural or synthetic materials. Exemplary natural materials include, but are not limited to, cellulose and modified cellulose, for example, diacetate or cellulose triacetate. Exemplary synthetic materials include, but are not limited to, polysulfone, polyvinyl, polyacetate, and the like. Said inert media can be obtained commercially or can be easily made by those skilled in the art. The binding partner can be attached to the inert medium by any means or method known to those skilled in the art including, for example, covalent conjugation. Alternatively, the binding partner can be associated with the inert matrix through the high-affinity, non-covalent interaction with an additional molecule which has been covalently attached to the inert medium. For example, a biotinylated linker pair may interact with avidin or streptavidin previously conjugated with the inert medium.

The absorbent matrix thus produced may be contacted with a biological fluid, or a fraction thereof, through the use of an extracorporeal circuit. The development and use of extracorporeal absorbent matrices has been extensively reviewed (see essler, Blood Purification 11: 150-157 (1993).

In a further aspect, referred to herein as the "stirred reactor" (Figure 2), the biological fluid can be exposed to the binding partner such as a mutein TNFα in a mixing chamber and, henceforth, the binding partner complex. The immune system inhibitor can be removed by means or methods known to those skilled in the art, including, for example, by mechanical, chemical or biological separation methods. For example, a mechanical separation method can be used in cases in which the binding partner, and therefore the binding partner / inhibitor complex of the immune system, represent the largest components of the biological fluid treated. In those cases, the leak can be used to retain the binding partner and the immune system inhibitors associated therewith, while allowing all other components of the biological fluid to permeate through the filter and, thus, be returned to the patient. In one example of a chemical or biological separation method, the binding partner and the immune system inhibitors associated therewith can be removed from the treated biological fluid through exposure to an absorbent matrix capable of specifically attaching to the binding partner. For example, a matrix constructed with antibodies reactive with a mutein TNFa can serve this purpose. Similarly, where the biotin conjugated to the binding partner such as a TNFa mutein prior to its addition to the biological fluid, a matrix constructed with avidin or streptavidin could be used to strip the binding partner and the immune system inhibitors associated with them from the binding partner. treated fluid. It is understood that removing the binding partner / inhibitor complex from the immune system, such as TNFα mutein bound to TNFR, from a biological fluid can be achieved by separating the biological fluid and the binding partner / inhibitor complex from the immune system in any appropriate manner. Either or both of the complex binding partner / inhibitor of the immune system and the biological fluid can be passively or actively separated from each other. Thus, for example, removing the TNFα mutein linked to TNFR from a biological fluid can be achieved by, for example, actively removing the TNFα mutein linked to TNFR from the biological fluid or actively removing the biological fluid from the TNFα mutein linked to TNFR.

In a final step of the present methods, the treated or altered biological fluid, which has a reduced amount of target inhibitors of the immune system such as the soluble TNF receptor, can be returned to the patient receiving treatment along with the untreated fractions of the fluid biological, if any of said fractions occurred during the treatment. The altered biological fluid can be administered to the mammal by any means or method known to those skilled in the art, including, for example, by direct infusion into the circulatory system. The altered biological fluid can be administered immediately after contact with the binding partner in a contemporary and extracorporeal circuit. In this circuit, the biological fluid can be (a) collected, (b) separated into cellular components and acellular, if desired, (c) exposed to the binding partner, and if necessary, separated from the binding partner linked to the target inhibitor of the immune system, (d) combined with the cellular component, if necessary, and (e) readmised the patient as altered biological fluid. In a further aspect, the altered acellular biological fluid can be administered to the patient at an infusion site different from the site where the cellular component of the biological fluid is administered to the patient. The administration of altered biological fluid to the patient can be simultaneous with, precede or follow the administration of the cellular component of the biological fluid to the patient. Alternatively, the administration of the altered biological fluid may be delayed under appropriate storage conditions determined by those skilled in the art.

If desired, the entire process can be repeated. Those skilled in the art can easily determine the benefits of repeated treatment by monitoring the clinical status of the patient and correlating that status with the concentrations of the target immune system inhibitor such as the circulating soluble TNFa receptor prior to, during and after treatment. .

In addition, an apparatus for reducing the amount of an objective inhibitor of the immune system such as the soluble TNF receptor in a biological fluid is provided. The apparatus may be composed of: (a) means for separating the biological fluid in a cellular component and an acellular component or a fraction thereof; (b) an absorbent matrix having attached thereto a TNFa mutein or a stirred reactor as described above to produce an altered acellular component or a fraction thereof and (c) means for combining the cellular fraction with the altered acellular component or fraction of it. The -aparate is particularly useful for whole blood as the biological fluid in which the cellular component separates either from the whole plasma or a fraction thereof.

The means for initially fractionating the biological fluid in the cellular component and the acellular component, or a fraction thereof, and for recombining the cellular component with the acellular component, or a fraction thereof, after the treatment are known to those skilled in the art. technique (see Apheresis: Principies and Practice, supra.).

An inhibitor of the immune system to be targeted may be sTNFRI (Seckinger et al., J. Biol. Quim 264: 1 1966-1 1973 (1989); Gatanaga et al., Proa Nati. Acad. Sci USA 87: 8781- 8784 (1990)), a naturally occurring inhibitor of the pluripotent immune system stimulator, TNF. The sTNFRI is produced by a proteolytic cleavage, which releases the extra-cellular domain of the tumor necrosis factor receptor of the membrane type I of its transmembrane and intracellular domains (Schall et al., CeH 61: 361-370 (1990 ); Himmler et al., DNA and Cell, Biol. 9: 705-715 (1990)). The sTNFRI retains the ability to bind to TNF with high affinity and, thus, inhibit the binding of TNF to the membrane receptor on the surfaces of cells.

The levels of sTNFRI in biological fluids increase in a variety of conditions that are characterized by an antecedent increase in TNF. These include bacterial, viral and parasite infections, and cancer as described above. In each of these disease states, the presence of the offending agent stimulates the production of TNF which stimulates a corresponding increase in the production of sTNFRI. The production of sTNFRI aims to reduce localized toxicity, as well. as systemic, associated with elevated levels of TNF and restore immune homeostasis.

In patients with tumors, the overproduction of sTNFRI can profoundly affect the course of the disease, considering the critical role of TNF in a variety of anti-tumor immune responses (reviewed in Beutler and Cerami, Ann. Rev. Immunol. 625-655 t (1989)). TNF directly induces the death of the fear cell by binding to the membrane-associated TNF receptor type I. In addition, the death of vascular endothelial cells is induced by the TNF binding, destroying the circulatory network that serves the tumor and also contributing to the death of the tumor cell. The critical roles for TNF in cytotoxic T-mediated cytolysis mediated by natural killer cell have also been documented. The inhibition of any or all of these effector mechanisms by sTNFRI has the potential to dramatically improve tumor survival.

The fact that sTNFRI promotes tumor survival and that removing it improves anti-tumor immunity has been demonstrated. In an experimental model of mouse tumor, it was found that the production of sTNFRI protects cells transformed in vitro from the cytotoxic effects of TNF and cytolysis mediated by natural killer cells and cytotoxic T lymphocytes (Selinsky et al, Immunol. 94: 88-93 (1998)). In addition, the secretion of sTNFRI by transformed cells has been shown to markedly improve its tumorigenicity and persistence in vivo (Selinsky and Howell, Cell, Immuno, 200: 81-87 (2000)). In addition, the removal of circulating sTNFRI has been shown to provide clinical benefit to cancer patients, as demonstrated by human Ultrapheresis tests as discussed above (Lentz, supra). These observations affirm the importance of this molecule in the survival of the tumor and suggest the development of methods to more specifically remove sTNFRI as new promising hopes for immunotherapy against cancer.

The following examples are given to provide those of ordinary skill in the art with a full disclosure and description of how the compounds, compositions, articles, devices and / or methods claimed in this document are made and evaluated and have the intention to be purely exemplary of the invention and do not intend to limit the scope of what the inventors consider as their invention. Efforts have been made to ensure accuracy with respect to numbers (eg, quantities, temperatures, etc.), but some errors and deviations must be taken into account. Unless otherwise indicated, the parts are parts by weight, the temperature is in ° C or at room temperature and the pressure is or is close to atmospheric.

EXAMPLE I Production, Purification and Characterization of the Immune System Inhibitor, Human sTNFRI The sTNFRI used in the present studies was produced recombinantly either in E. coli (R & D Systems, Minneapolis MN) or in eukaryotic cell culture as essentially described (See U.S. Patent No. 6,379,708, which is incorporated herein by reference). The construction of the eukaryotic expression plasma, the methods for the transformation and selection of cultured cells and for analyzing the production of sTNFRI by the transformed cells have been described (Selinsky et al., Supra, 1989).

The sTNFRI was detected and quantified in the present studies by capture ELISA (Selinsky et al, supra). In addition, the biological activity of the recombinant sTNFRI, that is, the ability to bind TNF, was confirmed by ELISA. The assay plates were covered with human TNFa (Quemicon, Temecula CA), blocked with bovine serum albumin and the sTNFRI, content in culture supernatants, as described above, was added. The bound sTNFRI was detected through the sequential addition of biotinylated goat anti-human sTNFRI, alkaline phosphatase conjugated streptavidin and p-nitrophenyl phosphate.

EXAMPLE 2 Production, Purification and Characterization of Mutains TNFa Briefly, the TNFa muteins 1, 2, 3 and 4 were produced by the expression of the respective cDNAs in E. coli. Genes coding for TNFα and TNFα muteins 1, 2, 3 and 4 were prepared using overlapping oligonucleotides having codons optimized for bacterial expression. Each of the coding sequences was fused in a frame with the one coding for the ompA leader to allow export of the recombinant polypeptides to the periplasm. The synthetic fragments were cloned with a pUC19 derivative immediately downstream with the Z lac promoter and the resulting recombinant plasmids were introduced into E. coli. The Recombinant bacterium was cultured late, induced with isopropyl ^ -D-thiogalactopyranoside (IPTG) for three hours and harvested by centrifugation. The periplasmic fractions were prepared and tested by ELISA using polyclonal goat antihuman TNFa capture antibodies. After the addition of the diluted periplasmas, TNFa and TNFa muteins bound 1, 2, 3 and 4 were detected by the sequential addition of biotinylated polyclonal goat anti-human TNFa, streptavidin-alkaline phosphate and para-nitrophenyl phosphate (pNPP). TNFa and each of the TNFa muteins were detectable in the respective periplasms, although the level of mutein TNFa 3 only slightly exceeded the detection limit of the analysis (Figure 4).

The TNFα and mutein TNFa 1, 2 and 4 polypeptides were purified from the periplasmic fractions by sequential chromatography on anion and cation exchange columns Q and S, respectively, essentially as described (Taverníer et al., J. Mol. Biol. 211: 493-501 (1990)). The TNFα and the mutein TNFα polypeptides were purified as > 95% homogeneity as analyzed by electrophoresis of sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE). The gels revealed a 17 kDa band corresponding to TNFa or muteins and a 34 kDa band, which was confirmed by the Western blot which was a dimerized TNFα mutein.

The TNFa muteins were tested for their ability to bind to sTNFRI. The wells of the microtiter plates were covered with TNFa, blocked and incubated with sTNFRI either in the presence or absence of the inhibitors, TNFa and TNFa muteins 1, 2 and 4. As shown in Figure 5, the TNFa 1 muteins , 2 and 4 are each linked to the sTNFRI.

EXAMPLE 3 Stripping of the Immune System Inhibitor, sTNFRI, from Human Plasma using Mutain Absorbing Matrices TNFa The absorptive matrices of TNFa muteins were produced and tested for their ability to strip sTNFRI from human plasma. Briefly, the purified TNFa muteins 1, 2 and 4 each were conjugated with cyanogenic bromide (CNBr) Sepharose ™ 4B at a density of 0.5 mg per mL of beads and the remaining CNBr groups were annealed with ethanolamine. The resulting matrices were packed in individual column housings and extensively washed with saline phosphate buffer before use.

Normal human plasma was reinforced (33% v / v) with culture supernatant containing recombinant human sTNFRI (see Example 1) for a final concentration of 8 nanograms per milliliter and passed through the respective columns at a flow rate of one milliliter of plasma per milliliter of resin per minute. An additional column, with protein not immobilized and tempered with ethanolamine, was included to control non-specific stripping. Fractions of one mL were collected and the relative levels of sTNFRI contained in the starting material and in the fractions were determined using a capture ELISA. To perform the capture ELISA, the wells were covered with polyclonal goat anti-sTNFRI and then blocked with 2% BSA. The plasma samples were diluted 1: 2, added to the wells and there the sTNFRI was captured. Biotinylated polyclonal goat anti-sTNFRI was added, followed by streptavidin-alkaline phosphatase and p-nitrophenyl phosphate. The relative absorbance at 405 nm was used to estimate stripping.

As shown in Figure 6, the three immobilized TNFα muteins effectively stripped the sTNFRI from human plasma and the hierarchy observed in Figure 5 re-manifested. The control matrix did not produce reduction in the sTNFRI levels, confirming the specificity of the deprivation observed with the mutein TNFa matrices. Importantly, the Quasi-quantitative stripping was achieved by the TNFα muteins 1 and 4 at a flow rate approaching that anticipated for use in a clinical setting.

EXAMPLE 4 Production and High Level Purification of Human Mutein TNFa 4 A high level expression system for the production of the human mutein TNFa 4 was developed. A human TNFα 4 mutein synthetic gene coding was produced by Blue Heron Biotechnology (Bothell, WA) from a series of overlapping oligonucleotides. The 5 'end of the gene contains a site for endonuclease restriction Nde I, which includes a methionine codon within the hexanucleotide recognition sequence. This methionine codon is fused in frame with the codons for six histidine residues (6His) and these are followed by additional codons that specify the seven amino acid recognition sequence released by tobacco attack virus protease (TEV). This recognition sequence is followed by a sequence encoding 157 amino acids representing the total extracellular portion of the native TNFα polypeptide, except for the substitution of tyrosine for serine at position 147. A stop codon within the frame follows the coding sequence of mutein TNFa and the stop codon is followed by a site for the restriction endonuclease Bam Hl. Except for the restriction endonuclease sites, the construct was optimized to reflect the E. coli codon tilt and to minimize the secondary structure of mRNA transcription. The synthetic gene was cloned into a derivative of pUC19 that is owned by Blue Heron and the nucleotide sequence of the coding region was confirmed in both strains using the dideoxy chain termination method.

The prokaryotic expression plasmid, pET-11a (EMD Biosciences, San Diego, CA), was used for the construction of a high level expression vector. The synthetic gene encoding the 6His-TEV-TNFa mutein was released from the recombinant plasmid based on pUC19 described above by digestion with Nde I and Bam Hl. The resulting fragment was purified by agarose gel electrophoresis and bound to unique A / de / and Bam Hl sites in pET-1 1a. The products of the ligand reaction were introduced by electroporation into the E. coli host strain, ABL21 (DE3) (EMD Biosciences). ABL21 (DE3) is a derivative of E. coli K 12, which contains a phage integration? lysogenic that has transduced the RNA polymerase of bacteriophage T7 in the bacterial genome. The inducible expression of the T7 polymerase gene allows high level transcription from the T7 promoter present immediately upstream of the mutein gene TNFa in pET-1 1 a. Stably transformed bacteria were selected in Luria Bertani plates containing 10 ug / ml of ampicillin. Individual colonies were selected based on the fidelity of the plasmid expression as assessed by the isolation of the recombinant plasmids and the release of the 6His-TEV-TNFa 4 mutein gene by combined digestion with Ndel and Bam Hl.

Cultures of ABL21 (DE3) that were transformed with the expression of the mutein TNFa 4 were cultured at 37 ° C, induced with IPTG and harvested by centrifugation. Bacterial granules were resuspended in lysis buffer and incubated for 30 minutes with DNAs and lysozyme. The lysate was homogenized and clarified by centrifugation. The TNFa 4 muteins contained within the lysate were purified using His Bind columns (EMD Biosciences) in accordance with the manufacturer's instructions. The purified TNFa 4 mutein was digested with TEV protease and the reaction mixture was rechromatographed on a His Bind column to remove the dissociated polyhistidine tag and the TEV protease., which also contains a polyhistidine tag. The purified TNFa 4 mutein was harvested in the His Bind column flow, dialyzed against buffered phosphate serum and stored at -20 ° C. The purity of the preparation was >95%, as evaluated with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).

EXAMPLE 5 In Vitro Stripping of sTNFRI and sTNFRIl from Plasma using an Extracorporeal Absorbent Device Constructed with Mutein TNFa 4 An extracorporeal absorbent device was produced sterile despite chemical conjugation of the purified TNFa 4 mutein to Actigel ALD Ultraflow 4 (Sterogene Bioseparations, Carlsbad, CA ). Actigel ALD Ultraflow is preferred for the construction of the absorbent device due to the superior stability of the aldehyde chemistry used for ligand conjugation and the proven stability and biocompatibility of cross-linked agarose beads in blood and plasma. The mutein TNFa 4 was conjugated to the beads at a density of 1 mg of mutein per mL of beads in accordance with the manufacturer's instructions. Sixty-two and a half mL of the matrix were packed by gravity flow in a polycarbonate housing (5 cm long x 4 cm in diameter), which contains 36 micron polypropylene filters at each end. The resulting device was washed sequentially with ten column volumes of 0.5 M NaCl, ten column volumes of 0.1 M glycine, pH 2.8 and ten column volumes of normal serum containing 0.1% sodium azide.

The mutein TNFa 4 absorber device was evaluated in vitro for its ability to strip human plasma sTNFR. Normal human plasma was filtered through a 0.45 micron filter and pumped through the device at a flow rate of thirty milliliters of plasma per minute. Thirty fractions of twenty-five mL were collected and the levels of human sTNFRI and sTNFRIl contained in the initial plasma sample and in the fractions were determined using capture ELISAs for sTNFRI and sTNFRIl. To perform the capture ELISAs, the wells were covered with either polyclonal goat anti-human sTNFRI or polyclonal goat anti-human sTNFRIl (R &D Systems) and then blocked with 2% BSA. The plasma samples were diluted 1: 2, added to the wells and then sTNFRI or sTNFRIl was captured therein. Biotinylated polyclonal goat anti-human sTNFRI or biotinylated polyclonal goat anti-human sTNFRIl (R &D Systems) were added, followed by streptavidin-alkaline phosphatase and p-nitrophenyl phosphate. The absorbance at 405 nm for each sample was used to calculate the concentration of sTNFRI and sTNFRIl relative to standard curves obtained using purified human sTNFRI and sTNFRIl (R & D Systems). The concentrations of human sTNFRI and sTNFRIl in the initial plasma sample were 1.17 ng / mL and 0.55 ng / mL, respectively. The sTNFRI and sTNFRIl were not detectable in any of the fractions collected after passing through the device, confirming the device's ability to strip both of these solutions of low plasma abundance.

The absorptive device of the mutein TNFa 4 was evaluated in an identical manner for its ability to strip the canine sTNFR from the plasma. Normal human plasma, previously stripped of the endogenous human sTNFRI and sTNFRIl by passing through the absorptive device of the mutein TNFa 4, was reinforced with sTNFRI and canine sTNFRIl at 2 ng / mL and 5 ng / mL, respectively. The canine sTNFR used in this analysis was recombinantly produced in cell cultures as described in Example 7 below. The reinforced plasma was filtered through a 0.45 micron filter and pumped through the device at a flow rate of thirty milliliters of plasma per minute. Twenty-five fractions of ten mL were collected and the levels of canine sTNFRI and sTNFRIl contained in the initial plasma sample and in the fractions were determined using capture ELISAs for sTNFRI and sTNFRIl. To perform the capture ELISAs, the wells were covered with either polyclonal goat anti-human sTNFRI or polyclonal goat anti-human sTNFRIl as above. After blocking them with 2% BSA, the plasma samples were diluted 1: 2 and added to the wells and the respective sTNFR in them were captured. Polyclonal rabbit IgG, reactive with the carboxy-terminal polyhistidine tag of sTNFRI and recombinant canine sTNFRI1, was added, followed by biotinylated anti-rabbit IgG, streptavidin-alkaline phosphatase and p-nitrophenyl phosphate. The absorbance at 405 nm for each sample was used to calculate the concentration of sTNFRI and sTNFRI1 relative to the standard curves obtained using purified recombinant canine sTNFRI and sTNFRIl. The concentrations of canine sTNFRI and sTNFRIl in the reinforced plasma sample, as evaluated with ELISA, were determined to be 2.8 ng / mL and 4.0 ng / mL, respectively. After passage through the absorbent device, the concentrations of sTNFRI in the plasma fractions were reduced from 57% to 82% and the sTNFRI1 was undetectable in any of the plasma fractions. These findings confirm the ability of the absorbent device of the human mutein TNFa 4 to also strip the canine sTNFR.

EXAMPLE 6 Ex vivo Stripping of Canine Plasma sTNFRI using an Extracorporeal Absorbent Device Constructed with Mutein TNFa 4. The ability of the absorptive device of the mutein TNFa 4 to strip the sTNFRI from the plasma, when used in an extracorporeal circuit not different from that used for the exchange of therapeutic plasma, was evaluated. Briefly, the blood was removed from a 47 kg dog through a double tube catheter implanted in the external jugular. The blood was delivered to Cobe Spectra, a centrifugal plasma separator, using a set of disposable Cobe Spectra Therapeutic Plasma Exchange tubes that had previously been sterilely modified by CytoLogic. Once separated, the blood cells and plasma were guided, independent of each other, through the rest of the system. The plasma component was passed through the mutein TNFa 4 absorber device as illustrated in Figure 1 of this document. The treated plasma was recombined with the blood cells and returned through the catheter to the animal. Various procedures, in which half to one volume of plasma was treated, were performed and each was completed in less than an hour.

The stripping of canine sTNFRI through this process was evaluated. Plasma samples were obtained from the extracorporeal circuit immediately before entering the absorbent device and immediately after removal from the absorbent device. The levels of canine sTNFRI in these plasma samples were determined using a capture ELISA. For To develop the capture ELISA, the wells were covered with monoclonal mouse anti-human sTNFRl (Becton-Dickinson, Mountain View, CA), previously shown to cross-react with a canine sTNFR1. The wells were then blocked with BSA of 2%. The plasma samples before and after the device were diluted 1: 2, added to the wells and the canine sTNFR1 was captured in it. Biotinylated polyclonal goat anti-human sTNFRl was added, followed by streptavidin-alkaline phosphatase and p-nitrophenyl phosphate. The absorbance at 405 nm for each sample was used to calculate the concentration of sTNFR1 relative to a standard curve obtained using purified recombinant canine sTNFR1. The pairs of plasma samples previous and ppsteriorés to the device, obtained during five separate treatments of this matter, were evaluated using this analysis (see the table below). In each of the sample pairs, a significant depletion of sTNFR1 was observed (range = 69.70% to 81.03 percent). The average percentage of sTNFR1 depletion for these five treatments was 74.90? 5.66.

EXAMPLE 7 Production and Purification of sTNFRl and Canine sTNFRIl The canine sTNFR used in these studies was produced in eukaryotic cell culture. The synthetic genes encoding canT sTNFR1 and sTNFRI1 were produced by Blue Heron Biotechnology of a series of overlapping oligonucleotides. The canine sTNFRI cDNA sequence was based on that contained in Genbank Accession Number XM_849381. The sequence of the canine sTNFRIl cDNA was based on that contained in Genbank Accession Number XM_544562. Each of the cDNAs was fused within the frame at the 3 'end for codons by six histidine residues (6 His) followed by a stop codon within the frame. The 5 'and 3' ends of each gene were flanked by recognition sequences for the restriction endonucleases Hind III and Xho I, respectively. Except for restriction endonuclease sites, each construct was optimized to reflect the codon deviation of Canis Familiaris and to minimize the secondary structure of mRNA transcription. Each synthetic gene was cloned with a derivative of pUC19 that is owned by Blue Heron and the nucleotide sequence of each coding region was confirmed in both strains using the dideoxy chain termination method.

The synthetic genes encoding canine sTNFRI and sTNFRIl were released from the recombinant pUC19-based plasmids described above by digestion with Hind III and Xho I. The resulting fragments were purified by agarose gel electrophoresis and ligated to unique sites of Hind III and Xho I in the eukaryotic expression vector, pCEP4 (Invitrogen, Carlsbad, CA). The products of the binding reactions were introduced by electroporation into the host strain of E. coli, ABL21 (EMD Biosciences). Stably transformed bacteria were selected in Luria Bertani plates containing 100 ug / mL of ampicillin. The individual colonies were selected based on the fidelity of the plasmid expression as assessed by the isolation of the recombinant plasmids and the release of the respective sTNFR genes by combined digestion with Hind III and Xho I.

Canine sTNFR expression plasmids were introduced into cell cultures Cf2Th (American Type Culture Collection, Manassas, VA) using lipofectamine. The cloned transfectant cell lines producing sTNFRI (Clone E) and sTNFRIl (Clone F) were isolated by limiting the solution in the presence of hygromycin (0.1 mg of drug active / ml). The cultures of Clone E and Clone F were cultured in shake flasks and recombinant canTNFRI and sTNFRIs, respectively, were purified by affinity chromatography on columns of human TNFα or human mutein TNFα 4 immobilized on Sepharose cyanide bromide. The purified sTNFRs were dialysed against PBS and stored at -20 ° C.

Although the invention has been described with reference to the currently preferred aspects, it should be understood that various modifications can be made without departing from the spirit of the invention. Accordingly, the invention is limited only by the following claims.

Claims (84)

1. CLAIMS 1. A conjugate comprising a tumor necrosis factor a (TNFa) mutein fixed to a substrate.
2. 2. The conjugate according to claim 1, wherein the mutein TNFa comprises the conserved sequence mentioned as SEQ ID NO: 1.
3. 3. The conjugate according to claim 1, wherein the mutein TNFa has the consensus sequence SEQ ID NO: 9, wherein Xi is an amino acid selected from Leu and Val, wherein X2 is a peptide of 2 or 3 amino acids having Gln or Arg in position 1, Asn, Ala or Thr in position 2 and Ser, Leu, Pro or absence in position 3, for example, selected from GlnAsnSer, ArgAlaLeu, ArgThrPro, GlnAlaSer and GlnThr, where X3 is a selected amino acid of Asp and Asn, wherein X4 is a 5 amino acid peptide having His, Pro, Leu, He or Val in position 1, Gln, Glu, Ser, Asn or Lys in position 2, Val, Ala or Be in position 3, Glu or Pro in position 4 and Glu or Gly in position 5, for example, selected from HisGlnValGIuGlu (SEQ ID NO: 21), HisGlnAlaGluGlu (SEQ ID NO: 22), ProGInValGIuGly (SEQ ID NO : 23), ProGluAlaGluGly (SEQ ID NO: 24), LeuSerAlaProGly (SEQ ID NO: 25), HeSerAlaProGly (SEQ ID NO: 26), ProGInAlaGluGly (SEQ ID NO: 27), NeAsnSerPr oGly (SEQ ID NO: 28) and ValLysAlaGluGly (SEQ ID NO: 29), wherein Xs is an amino acid selected from Glu, Gln and Arg, wherein Xe is a 4 amino acid peptide having Leu, Gly, Trp or Gln in position 1, Ser, Asp or Asn in position 2, Gln, Arg, Ser or Gly in position 3 and Arg or Tyr in position 4, for example, selected from LeuSerGInArg (SEQ ID NO: 30), LeuSerArgArg (SEQ ID NO: 31), GlyAspSerTyr (SEQ ID NO: 32), LeuSerGIyArg (SEQ ID NO: 33), TrpAspSerTyr (SEQ ID NO: 34), GlnSerGIyTyr (SEQ ID NO: 35) and LeuAsnArgArg (SEQ ID NO: 36), wherein X7 is an amino acid selected from Leu, Met and Lys, wherein? ß is a peptide of two amino acids having Met or Val in position 1 and Asp, Lys, Glu or GIn in position 2, for example, selected from MetAsp, MetLys, ValGIu, ValLys and ValGIn, where X9 is an amino acid selected from Lys, Thr, Glu and Arg, wherein X10 is an amino acid selected from Val, Lys e lie, where Xn is a 2 amino acid peptide having Ala, Ser, Thr or Leu at position 1 and Asp or Glu in position 2, for example, selected from AlaAsp, SerAsp, ThrAsp, LeuASp, AlaGlu and SerGIu, where Xí2 is an amino acid selected from Lys, Ser, Thr and Arg, where X13 is an amino acid selected from GIn and His, wherein X14 is a 4 or 5 amino acid peptide having Asp, Ser or Pro at position 1, Val, Tyr, Pro or Thr at position 2, Val, Pro, His or Asn at position 3, Leu or Val in position 4 and Leu, Phe or absence in position 5, for example, selected from AspValValLeu (SEQ ID NO: 37), AspTyrValLeu (SEQ ID NO: 38), SerTyrV alLeu (SEQ ID NO: 39), ProProProVal (SEQ ID NO: 40), SerThrHisValLeu (SEQ ID NO: 41), SerThrProLeuPhe (SEQ ID NO: 42) and SerThrAsnValPhe (SEQ ID NO: 43), where X15 is a selected amino acid of Val e lie, wherein X 6 is an amino acid selected from Phe, He and Leu, wherein X17 is an amino acid selected from He and Val, wherein X18 is a 2 amino acid peptide having GIn or Pro in the position 1 and Glu, Asn, Thr or Ser in position 2, for example, selected from GlnGlu, ProAsn, GlnThr and ProSer, wherein X 9 is an amino acid selected from Leu e He, where X20 is a 3 amino acid peptide having Pro, His or GIn at position, Lys, Arg or Thr at position 2 and Asp or Glu at position 3, for example, selected from ProLysAsp, HisArgGlu, GlnArgGlu and HisThrGIu, where X2i is an amino acid selected from Gly, Glu, GIn and Trp or is absent, wherein X22 is an amino acid selected from Leu, Pro and Ala, wherein X23 is an amino acid selected from Leu and GIn, wherein X2 is an amino acid selected from Gly and Asp, wherein X2s is an amino acid selected from Gln, Leu and Arg, wherein X26 is an amino acid selected from Ala and Thr, wherein X27 is an amino acid selected from Val e He, wherein X2s is an amino acid selected from Leu, Gln and Arg, wherein X2g is an amino acid selected from Lys, Glu, Ala, Asn and Asp, wherein X30 is an amino acid selected from Phe, He, Leu and Tyr and wherein X31 is an amino acid selected from Val e He.
4. 4. The conjugate according to claim 1, wherein the mutein TNFa has an amino acid substitution in a region of TNFa selected from region 1, amino acids 29-36, region 2, amino acids 84-91 and region 3, amino acids 143-149 , of human TNFα (SEQ ID NO: 2) or the analogous position of TNFα of other species.
5. 5. The conjugate according to claim 1, wherein the mutein TNFa is selected from mutein 1 (SEQ ID NO: 3), mutein 2 (SEQ ID NO: 4), mutein 3 (SEQ ID NO: 5), mutein 4 ( SEQ ID NO: 6), mutein 5 (SEQ ID NO: 7) and mutein 6 (SEQ ID NO: 8).
6. 6. The conjugate according to claim 1, wherein the mutein TNFa is selected from mutein 1 (SEQ ID NO: 3), mutein 2 (SEQ ID NO: 4) and mutein 4 (SEQ ID NO: 6).
7. 7. The conjugate according to claim 1, wherein the mutein TNFa is derived from a selected species of human, dog, cat, horse, sheep, goat, pig, cow, rabbit and rat.
8. 8. The conjugate according to claim 1, wherein the mutein TNFa is covalently bound to said substrate.
9. 9. The conjugate according to claim 1, wherein said substrate is an inert medium.
10. 10. The conjugate according to claim 9, wherein the inert medium is a hollow fiber.
11. 11. The conjugate according to claim 9, wherein the inert medium is in the form of an account.
12. 12. The conjugate according to claim 11, wherein the account is a macroporous account.
13. 13. The conjugate according to claim 12, wherein the macroporous bead is selected from agarose, cross agarose, cellulose, controlled pore glass, polyacrylamide, azlactone, polymethylacrylate and polystyrene.
14. 14. The conjugate according to claim 1, wherein the account is a non-porous account.
15. 15. The conjugate according to claim 14, wherein the non-porous bead is selected from silica, polystyrene and latex.
16. 16. The conjugate according to claim 9, wherein the inert medium is a cellulose-based fiber.
17. 17. The conjugate according to claim 9, wherein the inert medium is a synthetic fiber.
18. 18. The conjugate according to claim 9, wherein the inert medium is a flat or pleated membrane.
19. 19. The conjugate according to claim 9, wherein the inert medium is a silica-based particle.
20. 20. The conjugate according to claim 9, wherein the inert medium is an agarose-based particle.
21. 21. The conjugate according to claim 1, wherein the mutein TNFa is dimeric.
22. 22. The conjugate according to claim 21, wherein the dimeric TNFα mutein comprises two identical amino acid sequences.
23. 23. The conjugate according to claim 21, wherein the dimeric TNFα mutein comprises two non-identical amino acid sequences.
24. 24. The conjugate according to claim 1, wherein the mutein TNFa is monomeric.
25. 25. The conjugate according to claim 1, wherein the mutein TNFa has reduced TNF agonist activity relative to native TNFa.
26. 26. The conjugate according to claim 1, wherein the mutein TNFa has decreased signaling through the membrane receptors relative to native TNFa.
27. 27. The conjugate according to claim 1, wherein the mutein TNFa has decreased cytotoxic activity relative to native TNFa.
28. 28. The conjugate according to claim 1, wherein the mutein TNFa has decreased in vivo toxicity relative to native TNFa.
29. 29. The conjugate according to claim 1, wherein the mutein TNFa is derived from TNFa of the same species as that of the mammal.
30. 30. The conjugate according to claim 1, wherein the mutein TNFa has a reduced ability to form multimeric TNFa relative to native TNFa.
31. 31 A method for stimulating an immune response in a mammal having a pathological condition, comprising: (a) obtaining a biological fluid from a mammal, (b) contacting the biological fluid with a tumor necrosis factor a mutein ( TNFa) having specific binding activity for a soluble tumor necrosis factor (TNFR) receptor, (c) removing the TNFa mutein linked to said soluble TNFR from said biological fluid to produce an altered biological fluid having a reduced amount of TNFR soluble and (d) administer the altered biological fluid to the mammal.
32. 32. The method according to claim 31, wherein said altered biological fluid is selected from blood, plasma, serum and lymphatic fluid.
33. 33. The method according to claim 32, wherein the blood is whole blood.
34. 34. The method according to claim 33, further comprising the step of separating whole blood into a cellular component and an acellular component or a fraction of the acellular component, wherein said acellular component or acellular component fraction contains a soluble TNFR.
35. 35. The method according to claim 34, further comprising the step of combining the cellular component with the altered acellular component or altered fraction of the acellular component to produce altered whole blood.
36. 36. The method according to claim 31, wherein the mutein TNFa comprises the conserved sequence mentioned as SEQ ID NO: 1.
37. 37. The method according to claim 1, wherein the mutein TNFa has the consensus sequence SEQ ID NO: 9, wherein Xi is an amino acid selected from Leu and Val, wherein X2 is a peptide of 2 or 3 amino acids having Gln or Arg in position 1, Asn, Ala or Thr in position 2 and Ser, Leu, Pro or absence in position 3, for example, selected from GlnAsnSer, ArgAlaLeu, ArgThrPro, GlnAlaSer and GlnThr, where X3 is a selected amino acid of Asp and Asn, wherein X4 is a 5 amino acid peptide having His, Pro, Leu, Lie or Val in position 1, Gln, Glu, Ser, Asn or Lys n position 2, Val, Ala or Be in position 3, Glu or Pro in position 4 and Glu or Gly in position 5, for example, selected from HisGlnVaIGluGlu (SEQ ID NO: 21), HisGlnAlaGluGlu (SEQ ID NO: 22), ProGInVaIGluGly (SEQ ID NO : 23), ProGluAlaGluGly (SEQ ID NO: 24), LeuSerAlaProGly (SEQ ID NO: 25), HeSerAlaProGly (SEQ ID NO: 26), ProGInAlaGluGly (SEQ ID NO: 27), HeAsnSerProGl and (SEQ ID NO: 28) and ValLysAlaGluGly (SEQ ID NO: 29), wherein Xs is an amino acid selected from Glu, Gln and Arg, where? ß is a 4 amino acid peptide having Leu, Gly, Trp or GIn in position 1, Ser, Asp or Asn in position 2, GIn, Arg, Ser or Gly in position 3 and Arg or Tyr in position 4, for example, selected from LeuSerGInArg (SEQ ID NO: 30), LeuSerArgArg (SEQ ID NO: 31), GIyAspSerTyr (SEQ ID NO: 32), LeuSerGIyArg (SEQ ID NO: 33), TrpAspSerTyr (SEQ ID NO : 34), GlnSerGIyTyr (SEQ ID NO: 35) and LeuAsnArgArg (SEQ ID NO: 36), wherein X7 is an amino acid selected from Leu, Met and Lys, wherein Xa is a two amino acid peptide having Met or Val in position 1 and Asp, Lys, Glu or GIn in position '2, for example, selected from MetAsp, MetLys, ValGIu, ValLys and ValGIn, where X9 is an amino acid selected from Lys, Thr, Glu and Arg, in where X10 is an amino acid selected from Val, Lys e lie, where X is a 2 amino acid peptide having Ala, Ser, Thr or Leu in position 1 and Asp or Glu in Ta position 2, for example, selected from AlaAsp , You will p, ThrAsp, LeuASp, AlaGlu and SerGIu, where X 2 is an amino acid selected from Lys, Ser, Thr and Arg, where X13 is an amino acid selected from GIn and His, where X1 is a peptide of 4 or 5 amino acids that has Asp, Ser or Pro in position 1, Val, Tyr, Pro or Thr in position 2, Val, Pro, His or Asn in position 3, Leu or Val in position 4 and Leu, Phe or absence in position 5, for example, selected from AspValValLeu (SEQ ID NO: 37), AspTyrValLeu (SEQ ID NO: 38), SerTyrValLeu (SEQ ID NO: 39), ProProProVal (SEQ ID NO: 40), SerThrHisValLeu (SEQ ID NO: 41) , SerThrProLeuPhe (SEQ ID NO: 42) and SerThrAsnValPhe (SEQ ID NO: 43), where X15 is an amino acid selected from Val e He, where ?? 6 is an amino acid selected from Phe, He and Leu, where X -i7 is an amino acid selected from He and Val, wherein X 8 is a 2 amino acid peptide having GIn or Pro at position 1 and Glu, Asn, Thr or Ser at position 2, for example, selected from GlnGlu, ProAsn, GlnThr and ProSer, where Xig is an amino acid selected from Leu e He, wherein X20 is a 3 amino acid peptide having Pro, His or Gln at position 1, Lys, Arg or Thr at position 2 and Asp or Glu at position 3, for example, selected from ProLysAsp, HisArgGlu, GlnArgGlu and HisThrGIu, wherein X2i is an amino acid selected from Gly, Glu, Gln and Trp or absent, wherein X22 is an amino acid selected from Leu, Pro and Ala, wherein X23 is an amino acid selected from Leu and Gln, wherein X2 is an amino acid selected from Gly and Asp, wherein X25 is an amino acid selected from Gln, Leu and Arg, wherein X26 is an amino acid selected from Ala and Thr, wherein X27 is an amino acid selected from Val e lie, where X28 is an amino acid selected from Leu, Gln and Arg, wherein X29 is an amino acid selected from Lys, Glu, Ala, Asn and Asp, wherein X30 is an amino acid selected from Phe, Me, Leu and Tyr and wherein X31 is a selected amino acid from Val e lie.
38. 38. The method according to claim 31, wherein the mutein TNFα has an amino acid substitution in a region of TNFα selected from region 1, amino acids 29-36, region 2, amino acids 84-91 and region 3, amino acids 143-149 , of human TNFα (SEQ ID NO: 2) or the analogous position of TNFα of other species.
39. 39. The conjugate according to claim 31, wherein the mutein TNFa is selected from mutein 1 (SEQ ID NO: 3), mutein 2 (SEQ ID NO: 4), mutein 3 (SEQ ID NO: 5), mutein 4 ( SEQ ID NO: 6), mutein 5 (SEQ ID NO: 7) and mutein 6 (SEQ ID NO: 8).
40. 40. The conjugate according to claim 31, wherein the mutein TNFa is selected from mutein 1 (SEQ ID NO: 3), mutein 2 (SEQ ID NO: 4) and mutein 4 (SEQ ID NO: 6).
41. 41. The method according to claim 31, wherein said mutein TNFa has specific binding activity for a single type of soluble TNFR.
42. 42. The method according to claim 41, wherein said soluble TNFR is sTNFRI.
43. 43. The method according to claim 41, wherein said soluble TNFR is sTNFRII.
44. 44. The method according to claim 31, wherein the biological fluid is contacted with a TNFa mutein that has specific binding activity for more than one type of soluble TNFR.
45. 45. The method according to claim 44, wherein said mutein TNFa has specific binding activity for sTNFRI and sTNFRII.
46. 46. The method according to claim 31, wherein the mutein TNFa is fixed to an inert medium to form an absorbent matrix.
47. 47. The method according to claim 46, wherein the mutein TNFa is covalently fixed to the inert medium:
48. 48. The method according to claim 46, wherein the inert medium is a hollow fiber.
49. 49. The method according to claim 46, wherein the inert medium is a macroporous count.
50. 50. The method according to claim 46, wherein the inert medium is a fiber with cellulose base.
51. 51. The method according to claim 46, wherein the inert medium is a synthetic fiber.
52. 52. The method according to claim 46, wherein the inert medium is a flat or pleated membrane.
53. 53. The method according to claim 46, wherein the inert medium is a silica-based particle.
54. 54. The method according to claim 31, wherein said mutein TNFa is produced recombinantly.
55. 55. The method according to claim 31, wherein the biological fluid is contacted with a plurality of TNFa muteins.
56. 56. The method according to claim 55, wherein said plurality of TNFa muteins has specific binding activity for a single type of soluble TNFR.
57. 57. The method according to claim 56, wherein said soluble TNFR is sTNFRI.
58. 58. The method according to claim 56, wherein the soluble TNFR is sTNFRII.
59. 59. The method according to claim 31, wherein the biological fluid is contacted with a plurality of TNFα muteins that have specific binding activity for more than one type of soluble TNFR.
60. 60. The method according to claim 59, wherein said plurality of TNFa muteins has specific binding activity for sTNFRI and sTNFRII.
61. 61 / The method according to claim 31, wherein the mutein TNFα is conjugated to a carrier.
62. 62. The method according to claim 59, wherein said plurality of mutein TNFa is conjugated to a carrier.
63. 63. The method according to claim 31, wherein steps (a) to (d) are repeated.
64. 64. The method according to claim 31, wherein the mammal is a human.
65. 65. The method according to claim 31, wherein the mammal is not a human.
66. 66. The method according to claim 31, wherein the mutein TNFα linked to the soluble TNFR is removed by mechanical methods.
67. 67. The method according to claim 31, wherein the mutein TNFα linked to soluble TNFR is removed by chemical or biological methods.
68. 68. The method according to claim 31, wherein the mutein TNFa bound to the soluble TNFR is removed by separating the biological fluid from the mutein TNFa.
69. 69. A method for stimulating an immune system response in a mammal having a pathological condition, comprising: (a) obtaining a biological fluid from a mammal, (b) contacting the biological fluid with a necrosis factor-a mutein. tumor (TNFa) having specific binding activity for a soluble tumor necrosis factor receptor (TNFR), wherein the mutein TNFa is fixed to an inert medium to form an absorbent matrix, (c) removing the absorbent matrix comprising the mutein TNFa linked to said soluble TNFR of said biological fluid to produce an altered biological fluid and (d). administer the altered biological fluid to the mammal.
70. 70. The method according to claim 69, wherein said biological fluid is selected from blood, plasma, serum and lymphatic fluid.
71. 71. The method according to claim 70, wherein the blood is whole blood.
72. 72. The method according to claim 71, further comprising the step of separating whole blood into a cellular component and an acellular component or a fraction of the acellular component, wherein said acellular component or said acellular component fraction contains a soluble TNFR.
73. 73. The method according to claim 72, further comprising the step of combining the cellular component with the altered acellular component or altered fraction of the acellular component to produce altered whole blood.
74. 74. A method for stimulating an immune response in a mammal having a pathological condition comprising: (a) obtaining a biological fluid from a mammal, (b) contacting the biological fluid with two or more TNFα muteins having specific binding activity for a soluble tumor necrosis factor receptor, (c) isolating the TNFα muteins linked to the soluble TNFR from the biological fluid to produce an altered biological fluid, and (f) administering the altered biological fluid to the mammal.
75. 75. The method according to claim 74, wherein two or more muteins TNFa are fixed to an inert medium to form an absorbent matrix.
76. 76. The method according to claim 75, wherein the two or more TNFa muteins are covalently bound to the inert medium.
77. 77. The method according to claim 74, wherein said biological fluid is selected from blood, plasma, serum and lymphatic fluid.
78. 78. The method according to claim 77, wherein the blood is whole blood.
79. 79. The method according to claim 78, further comprising the step of separating whole blood into a cellular component and an acellular component or a fraction of the acellular component, wherein said acellular component or said acellular component fraction contains a soluble TNFR.
80. 80. The method according to claim 79, further comprising the step of combining the cellular component with the altered acellular component or altered fraction of the acellular component to produce altered whole blood.
81. 81. A method for removing the soluble tumor necrosis factor (TNFR) receptor from the biological fluid, comprising: (a) obtaining a biological fluid, (b) contacting the biological fluid with a tumor necrosis factor a mutein (TNFa) having specific binding activity for a soluble TNFR, (c) removing the TNFα mutein linked to said soluble TNFR from said biological fluid to produce an altered biological fluid having a reduced amount of soluble TNFR.
82. 82. The method according to claim 81, wherein the mutein TNFa is fixed to an inert medium to form an absorbent matrix, wherein the mutein TNFa linked to said soluble TNFR of said biological fluid upon removal of the absorbent matrix comprising the mutein TNFa linked to the soluble TNFR of the biological fluid.
83. 83. The method according to claim 81, wherein in step (b) the biological fluid is also contacted with one or more additional TNFa muteins having specific binding activity for a soluble TNFR, wherein in step (c) one or more additional TNFa muteins linked to said soluble TNFR are also removed from said soluble fluid.
84. 84. The method according to claim 81, wherein the biological fluid is contacted with a plurality of TNFa muteins.