WO2008127515A1

WO2008127515A1 - Compositions and method for enhancing immune responses in mammals

Info

Publication number: WO2008127515A1
Application number: PCT/US2008/002737
Authority: WO
Inventors: Mark Douglas Howell
Original assignee: Cytologic, Inc.
Priority date: 2007-03-01
Filing date: 2008-02-29
Publication date: 2008-10-23

Abstract

The present invention provides a method for enhancing an immune response in a mammal to facilitate the elimination of a chronic pathology. The method involves the neutralization of immune system inhibitors such as soluble TNF receptor in the circulation of the mammal, thus, enabling a more vigorous immune response to the pathogenic agent. The neutralization of immune system inhibitors is accomplished by contacting biological fluids of a mammal with one or more binding partners) capable of binding to and, thus, neutralizing the targeted immune system inhibitor(s) in the biological fluids. Particularly useful in the invention are TNFα muteins and dimeric fusion proteins of TNFα or TNFα muteins that bind soluble TNFR with affinities sufficient to neutralize the biological activity of the soluble TNFR and display reduced binding to, or signaling through, membrane TNFR relative to wild type TNFαthus, reducing or eliminating toxicity.

Description

COMPOSITIONS AND METHOD FOR ENHANCING IMMUNE RESPONSES IN

MAMMALS

CROSS-REFERENCE TO RELATED APPLICATIONS

[001] This application claims priority to U.S. provisional application number 60/904,261 filed on March 1, 2007. The aforementioned application is herein incorporated by this reference in its entirety.

[002] This invention relates generally to the field of immunotherapy and, more specifically, to methods for enhancing host immune responses.

BACKGROUND OF THE INVENTION

[003] The immune system of mammals has evolved to protect the host against the growth and proliferation of potentially deleterious agents. These agents include infectious microorganisms such as bacteria, viruses, fungi, and parasites which exist in the environment and which, upon introduction to the body of the host, can induce varied pathological conditions. Other pathological conditions may derive from agents not acquired from the environment, but rather which arise spontaneously within the body of the host. The best examples are the numerous malignancies known to occur in mammals. Ideally, the presence of these deleterious- agents in a host triggers the mobilization of the immune system to effect the destruction of the agent and, thus, restore the sanctity of the host environment.

[004] 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 immunity. The first line of defense is mediated by the mechanisms of innate immunity. Innate immunity does not discriminate among the myriad agents that might gain entry into the host's body. Rather, it responds in a generalized manner that employs the inflammatory response, phagocytes, and plasma-borne 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 it serves, in large, part, to amplify and focus the effector mechanisms of innate immunity.

[005] The elaboration of an effective immune response requires contributions from both innate and specific immune mechanisms. The function of each of these arms of the immune system individually, as well as their interaction with each other, is carefully coordinated, both in a temporal/spatial manner and in terms of the particular cell types that participate. This coordination results from the actions of a number of soluble immunostimulatory mediators or "immune system stimulators" (reviewed in, Trinchieri et al., J. Cell, Biochem. 53:301-308 (1993)). Certain of these immune system stimulators initiate and perpetuate the inflammatory response and the attendant systemic sequelae. Examples of these include, but are not limited to, the proinflammatory mediators tumor necrosis factors a and β, interleukin 1 , interleukin- 6, interleukin-8, interferon-γ, and the chemokines RANTES, macrophage inflammatory proteins 1-α and l-β and macrophage chemotactic and activating factor. Other immune system stimulators facilitate 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-γ. Still other immune system stimulators mediate bidirectional communication between specific immunity and innate immunity. Examples of these include, but are not limited to, interferon-7, interleukin- 1, tumor necrosis factors a and /3, and interleukin 12. All of these immune system stimulators exert their effects by binding the specific receptors on the surface of host cells, resulting in the delivery of intercellular signals that alter the function of the target cell. Cooperatively, these mediators stimulate the activation and proliferation of immune cells, recruit them to particular anatomical sites, and permit their collaboration in the elimination of the offending agent. The immune response induced in any individual is determined by the particular complement of immune system stimulators produced, and by the relative abundance of each.

[006] 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. IM. Med. 40:365-394 (1995)). These "immune system inhibitors" provide the immune system with the ability to dampen responses in order to prevent the establishment of a chronic inflammatory state with the potential to damage the host's tissues. Regulation of host immune function by immune system inhibitors is accomplished through a variety of mechanisms as described below.

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

[008] Second, certain immune system inhibitors antagonize the binding of immune system stimulators to their receptors. By way of example, 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, by virtue of occupying the interleukin-1 membrane receptor, blocks the effects of interleukin-1.

[009] Third, particular immune system inhibitors exert their effects by binding to receptors on host cells and signaling a decrease in their production of immune system stimulators. Examples include, but are not limited to, interferon-/?, which decreases the production of two key proinflammatory mediators, tumor necrosis factor a and interleukin- 1 (Coclet-Ninin et al., Eur. Cytokine Network 8:345349 (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 immune system stimulators, certain immune system inhibitors also enhance the production of other immune system inhibitors. By way of example, interferon-an inhibits interleukin-1 and tumor necrosis factor a production and increases the production of the corresponding immune system inhibitors, interleukin-1 receptor antagonist and soluble receptors for tumor necrosis factors αand β fPinarello. Sem. in Oncol. 24(3 Suppl. 9):81-93 (1997).

[010] Fourth, certain immune system inhibitors act directly on immune cells, inhibiting their proliferation and function, thereby decreasing the vigor of the immune response. By way of example, transforming growth factor-/? 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 immunosuppressive mechanisms are intended to regulate the immune response, both quantitatively and qualitatively, to minimize the potential for collateral damage to the host's own tissues.

[011] In addition to the inhibitors produced by the host's immune system for self-regulation, other immune system inhibitors are produced by infectious microorganisms. For example, many viruses produce molecules which are viral homologues of 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, tumor necrosis factors a and /3, and interferons a, β, and γ. Similarly, helminthic parasites produce homologues of host immune system inhibitors (reviewed in Riffkin et al., Immunol. Cell Biol. 74:564-574 (1996)), and several bacterial genera are known to produce immunosuppressive products (reviewed in, Reimann et al., Scand. J. Immunol. 31 :543-546 (1990)). All of these immune system inhibitors serve to suppress the immune response during the initial stages of infection, to provide advantage to the microbe, and to enhance the virulence and chronicity of the infection.

[012] A role for host-derived immune system inhibitors in chronic disease also has been established. In the majority of cases, this reflects a polarized T cell response during the initial infection, wherein the production of immunosuppressive mediators (i.e., interleukin-4, interleukin-10, and/or transforming growth factor-(jS) dominates over the production of immunostimulatory mediators (i.e., interleukin-2, interferon-γ, and/or tumor necrosis factor β) (reviewed in Lucey et al., Clin, Micro. Rev. 9:532-562 (1996)). Overproduction of immunosuppressive mediators of this type has been shown to produce chronic, non-healing pathologies in a number of medically important diseases. These include, but are not limited to, diseases resulting from infection with: 1) the parasites, Plasmodium falciparum (Sarthou et al.. Infect. Immun. 65:3271-3276 (1997)), Trypanosoma cruzi (reviewed in Laucella et al., Revista Argentina de Microbiolgia 28:99-109 (1996)), Leishmania major (reviewed in Etges and Muller, J. MoI. Med. 76:372-390 (1998)), and certain helminths (Riffkin et al., supra); 2) the 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: l 130-1133 (1995)), and Listeria monocytogenes (Blauer et al., J. Interferon Cytokine Res. 15: 105-114 (1995)), and, 3) intracellular fungus, Candida albicans (reviewed in Romani et al., Immunol. Res. 14: 148-162 (1995)). The inability to spontaneously resolve infection is influenced by other host-derived immune system inhibitors as well. By way of example, interleukin-1 receptor antagonist and the soluble receptors for tumor necrosis factors a. and β are produced in response to interleukin-1 and tumor necrosis factor a. and/or β production driven by the presence of numerous infectious agents. Examples include, but are not limited to, infections by Plasmodium falciparum (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, Infect. Immun. 65:2100-2106 (1997)). In cases where the production of any of the aforementioned immune system inhibitors, either individually or in combination, dampens or otherwise alters immune responsiveness before the elimination of the pathogenic agent, a chronic infection may result.

[013] In addition to this role in infectious disease, host-derived immune system inhibitors contribute also to chronic malignant disease. Compelling evidence is provided by studies of soluble tumor necrosis factor receptor Type I (sTNFRI) in cancer patients. Nanomolar concentrations of sTNFRI are synthesized by a variety of activated immune cells in cancer patients 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, circulating sTNFRI levels often are elevated significantly in cancer patients (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 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, correlate with poorer treatment outcomes (Aderka et al., supra). These observations suggest that sTNFRI aids tumor survival by inhibiting anti-tumor immune mechanisms which employ tumor necrosis factors Oi and/or β (TNF), and they argue favorably for the clinical manipulation of sTNFRI levels as a therapeutic strategy for cancer.

[014] Direct evidence that the removal of immune system inhibitors provides clinical benefit derives from the evaluation of Ultrapheresis, a promising experimental cancer therapy (Lentz, J. Biol. Response Modif. 8:51 1 -527 (1989); Lentz. Trier. Apheresis 3:40-49 ( 1999): Lentz, Jpn. J. Apheresis 16:107-114 (1997)). Ultrapheresis involves extracorporeal fractionation of plasma components by ultrafiltration. Ultrapheresis selectively removes plasma components within a defined molecular size range, and it has been shown to provide significant clinical advantage to patients presenting with a variety of tumor types. Ultrapheresis induces pronounced inflammation at tumor sites, often in less than one hour post-initiation. This rapidity suggests a role for preformed chemical and/or cellular mediators in the elaboration of this inflammatory response, and it reflects the removal of naturally occurring plasma inhibitors of that response. Indeed, immune system inhibitors of TNFa and β, interleukin-1, and interleukin-6 are removed by Ultrapheresis (Lentz, Ther. Apheresis 3:40-49 (1999)). Notably, the removal of sTNFRI has been correlated with the observed clinical responses (Lentz, Ther. Apheresis 3:40-49 (1999); Lentz, Jpn. J. Apheresis 16:107- 114 (1997)).

[015] Ultrapheresis is in direct contrast to more traditional approaches which have endeavored to boost immunity through the addition of immune system stimulators. Preeminent among these has been the infusion of supraphysiological levels of TNF (Sidhu and Bollon, Pharmacol. Ther. 57:79-128 (1993)); and of interleukin-2 (Maas et al., Cancer Immunol. Immunother. 36: 141-148 (1993)), which indirectly stimulates the production of TNF. These therapies have enjoyed limited success (Sidhu and Bollon, StψϊU, Maas et al.,

SUpra) due to the fact: 1) that at the levels employed they proved extremely toxic; and 2) that each increases the plasma levels of the immune system inhibitor, sTNFRI (Lantz et al., Cytokine 2:402-406 (1990); Miles et al., Brit. J. Cancer 66: 1 195-1199 (1992)). Together, these observations support the utility of Ultrapheresis as a biotherapeutic approach to cancer - one which involves the removal of immune system inhibitors, rather than the addition of immune system stimulators. [016] Although Ultrapheresis provides advantages over traditional therapeutic approaches, there are certain drawback that limit its clinical usefulness. Not only are immune system inhibitors removed by Ultrapheresis, but other plasma components, including beneficial ones, are removed since the discrimination between removed and retained plasma components is based solely on molecular size. An additional drawback to Ultrapheresis is the significant loss of circulatory volume during treatment, which must be offset by the infusion of replacement fluid. The most effective replacement fluid is an ultrafiltrate produced, in an identical manner, from the plasma of non-tumor bearing donors. A typical treatment regimen (15 treatments, each with the removal of approximately 7 liters of ultrafiltrate) requires over 200 liters of donor plasma for the production of replacement fluid. The chronic shortage of donor plasma, combined with the risks of infection by human immunodeficiency virus, hepatitis A, B, and C or other etiologic agents, represents a severe impediment to the widespread implementation of Ultrapheresis.

[017] Because of the beneficial effects associated with the removal of immune system inhibitors using extracorporeal technologies, there exists a need for methods which can be used to specifically neutralize those inhibitors in situ. The present invention satisfies these needs and provides related advantages as well.

SUMMARY OF INVENTION

[018] The present invention provides a method for stimulating immune responses in a mammal through the neutralization of immune system inhibitors such as soluble TNF receptors present in the circulation of the mammal. The neutralization of immune system inhibitors such as soluble TNF receptors can be effected by contacting biological fluids from the mammal with a binding partner capable of selectively neutralizing the targeted immune system inhibitor, for example, TNFα muteins.

[019] Binding partners useful in these methods are TNFα muteins having specificity for soluble TNF receptors. Moreover, mixtures of TNFα muteins having specificity for one or more soluble TNF receptors can be used.

[020] In a particularly useful embodiment, the binding partner, such as a TNFα mutein, is administered intravenously to the mammal. The dose and schedule of said administration are parameters individualized for each patient, guided by the induction of vigorous immune responses while minimizing toxicity.

BRIEF DESCRIPTION OF THE FIGURES

[021] Figure 1 A shows an alignment of TNFα sequences from various mammalian species (mouse, SEQ ID NO: 10; rat, SEQ ID NO.l 1; 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 top sequence shows the conserved amino acids across the shown species (SEQ ID NO: l)(completely conserved or with one exception). Non-conserved amino acids are indicated by "." (taken from Van Ostade et al, Prot. Eng. 7:5-22 (1994), which is incorporated herein by reference). Figure IB shows an alignment of the conserved TNFα sequence with human TNFa and six representative TNFα muteins, designated 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). The four muteins differ from the human sequence by single amino acid substitutions, indicated with bold and underline. Figure IC shows a representative consensus TNFa sequence (SEQ ID NO:9).

[022] Figure 2 shows the presence of human TNFα and TNFα muteins 1, 2, 3 and 4 in periplasmic preparations of E sdoeruhia coli transformed with the respective expression constructs.

[023] Figure 3 shows that TNFα muteins bind to sTNFRI and inhibit its binding to TNFα. Wells of a microtiter plate were coated with TNFα, blocked, and incubated with sTNFRI either in the presence or absence of the inhibitors, TNFα and TNFα muteins 1 , 2 and 4.

DETAILED DESCRIPTION OF THE INVENTION

[024] The present invention provides methods to neutralize immune system inhibitors such as soluble TNF receptors in the circulation of a host mammal, thereby potentiating an immune response capable of resolving a pathological condition or decreasing the severity of a pathological condition. By enhancing the magnitude of the host's immune response, the methods of the present invention avoid the problems associated with the repeated administration of chemotherapeutic agents which often have undesirable side effects, for example, chemotherapeutic agents used in treating cancer.

[025] The present invention provides a method for enhancing an immune response in a mammal to facilitate the elimination of a chronic pathology. The method involves the neutralization of immune system inhibitors such as soluble TNF receptor in the circulation of the mammal, thus, enabling a more vigorous immune response to the pathogenic agent. The neutralization of immune system inhibitors is accomplished by contacting biological fluids of a mammal with one or more binding partner(s) capable of binding to and, thus, neutralizing the targeted immune system inhibitor(s) in the biological fluids. Particularly useful in the invention are TNFαmuteins and dimeric fusion proteins of TNFα or TNFa muteins: 1) which bind soluble TNFR with affinities sufficient to neutralize the biological activity of the soluble TNFR; and, 2) which display reduced binding to, or signaling through, membrane TNFR relative to wild type TNFα thus, reducing or eliminating toxicity.

[026] As used herein, the term "immune system stimulator" refers to soluble mediators that increase the magnitude of an immune response, or which encourage 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 proinflammatory mediators tumor necrosis factors a and /3, interleukin-1, interleukin-2, interleukin-4, interleukin-5, interleukin-6, interleukin-8, interleukin-12, interferon-γ, interferon-7; and the chemokines RANTES, macrophage inflammatory proteins 1-α and 1- β and macrophage chemotactic and activating factor, as discussed above.

[027] As used herein, the term "immune system inhibitor" refers to a soluble mediator that decreases the magnitude of an immune response, or which discourages the development of particular immune mechanisms that are more effective in resolving a specific pathological condition, or which encourages the development of particular immune mechanisms that are less effective in resolving a specific pathological condition. Examples of host-derived immune system inhibitors include interleukin-1 receptor antagonist, transforming growth factor-/?, interleukin-4, interleukin-10, or the soluble receptors for interleukin-1, interleukin-2, interleukin-4, interleukin-6, interleukin-7, interferon-7 and tumor necrosis factors a and β. In a particular embodiment of the present invention, the immune system inhibitor is soluble TNF receptor Type I (sTNFRI) or Type II (sTNFRII). Immune system inhibitors produced by microorganisms are also potential targets including, for example, homologues of the mammalian soluble receptors for tumor necrosis factor a and β. As used herein, the term "targeted" immune system inhibitor refers to that inhibitor, or collection of inhibitors, which is to be neutralized in the biological fluid by a method of the invention, for example, sTNFRI and/or sTNFRII.

[028] As used herein, the term "soluble TNF receptor" refers to a soluble form of a receptor for αand β. Two forms of TNF receptor have been identified, type I receptor (TNFRI), also known as TNF-R55, and type II receptor (TNFRII), also known as TNF-R75, both of which are membrane proteins that bind to a and β and mediate intracellular signaling. Both of these receptors also occur in a soluble form. The soluble form of TNF receptor functions as an immune system inhibitor, 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 of the invention, the methods can be used to neutralize one or both types of TNF receptor depending on whether the TNFα mutein or plurality of muteins used in the method binds to one or both types of receptors.

[029] As used herein, the term "mammal" can be a human or a non-human animal, such as dog, cat, horse, cattle, pig, sheep, non-human primate, mouse, rat, rabbit, or other mammals, for example. The term "patient" is used synonymously with the term "mammal" in describing the invention.

[030] 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 of or contributes to a disease state. Examples of such pathological conditions include, but are not limited to those resulting from persistent viral, bacterial, parasitic, and fungal infections, and cancer. Among individuals exhibiting such chronic diseases, those in whom the levels of immune system inhibitors are elevated are particularly suitable for the treatment of the invention. Plasma levels of immune system inhibitors can be determined using methods well known in the art (see, for example, Adolf and Apfler, SUpYd, 1991). Those skilled in the art readily can determine pathological conditions that would benefit from the depletion of immune system inhibitors according to the present methods.

[031] As used herein, the term "biological fluid" refers to a bodily fluid obtained from a mammal, for example, blood, including whole blood, plasma, serum, lymphatic fluid, or other types of bodily fluids.

[032] As used herein, the term "selectively binds" means that a molecule binds to one type of target molecule, but not substantially to other types of molecules. The term "specifically binds" is used interchangeably herein with "selectively binds."

[033] As used herein, the term "binding partner" is intended to include any molecule chosen for its ability to selectively bind to the targeted immune system inhibitor. The binding partner can be one which naturally binds the targeted immune system inhibitor. For example, tumor necrosis factor a and β can be used as a binding partner for sTNFRI. Alternatively, other binding partners, chosen for their ability to selectively bind to the targeted immune system inhibitor, can be used. These include fragments of the natural binding partner, polyclonal or monoclonal antibody preparations or fragments thereof, or synthetic peptides. In a particular embodiment of the present invention, the binding partner is a TNFα mutein.

[034] As used herein, the term "TNFa mutein" refers to a TNFo; variant having one or more amino acid substitutions relative to a parent sequence and retaining specific binding activity for a TNF receptor, either soluble and/or membrane TNFR. Generally, the muteins of the present invention have a single amino acid substitution relative to a parent sequence. Exemplary TNFo; muteins include the human TNFα muteins designated muteins 1, 2, 3, 4, 5 and 6 (see Figure IB), which are derived from human TNFαbut have a single amino acid substitution relative to the wild type sequence, as discussed below. It is understood that analogous muteins of species other than human are similarly included, for example, muteins analogous to muteins 1 , 2, 3, 4, 5 or 6 in the other mammalian species shown in Figure IA, or other mammalian species. These and other muteins, as described in more detail below, are included within the meaning of a TNFa mutein of the invention. [035] The present invention provides compositions and methods for neutralizing soluble TNFR or stimulating or enhancing an immune response in a mammal. The invention advantageously uses ligands that bind to immune system inhibitors to counterbalance the dampening effect of immune system inhibitors on the immune response. Such ligands are also referred to herein as "binding partners."

[036] A binding partner particularly useful in the present invention is a ligand that binds with high affinity to an immune system inhibitor, for example, soluble TNF receptor and in particular sTNFRI. Another useful characteristic of a binding partner is a lack of direct toxicity. For example, a binding partner lacking TNF agonist activity is particularly useful. It is advantageous to use a ligand that has affinity for an immune system inhibitor but has decreased ability to stimulate a biological response, that is, has decreased or low agonist activity. In this case, the ligand exhibits low biological activity with respect to membrane receptor signaling.

[037] Yet another useful characteristic of a binding partner is a lack of indirect toxicity, for example, immunogenicity. Because the binding partner is administered to the patient, an immune response against the ligand can be stimulated if the ligand is immunogenic, resulting in undesirable immune responses, particularly in a patient in which the method is being repeated. Therefore, a ligand having low immunogenicity would minimize any undesirable immune responses against the ligand. As disclosed herein, a particularly useful ligand to be used as a binding partner of the invention is derived from the same species as the patient being treated. For example, for treating a human, a human TNFα mutein can be used as the binding partner, which is expected to have low immunogenicity given the similarity to the endogenous TNFα. Similarly, muteins derived from other mammalian species can be used in the respective species.

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

[039] There are several advantages to using TNFα muteins as binding partners in the present invention. Although TNFα muteins can display lower binding activity for TNF receptors, some TNFα muteins bind only 5- to 17-fold less effectively than native TNFa. Such a binding affinity, albeit reduced relative to native TNFα, 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, for example, 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 though the binding affinity is reduced 10- to 17-fold, there can be a 200- to 10,000-fold decrease in signaling through membrane receptors, for example, decreased cytotoxic activity or in vivo toxicity. As discussed above, such a reduced signaling through membrane receptors, for example, reduced cytotoxicity or in vivo toxicity, is advantageous since the binding partner is administered to the patient.

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

[041] As disclosed herein, particularly useful muteins are human muteins 1, 2, 3, 4, 5 and 6 (Figure 1 B) and the analogous muteins in other mammalian species. Mutein 1 is a single amino acid substitution relative to wild type human TNFα of Arg³¹ with Pro (Zhang et al., s upra , 1992). This mutein exhibits approximately 10-fold lower binding activity to 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 TNFα 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 Pro^w with Leu (Yamagishi et al., supra, 1990; Pro 1 15 in the numbering system of Yamagishi et al.). This mutein exhibits approximately 12-fold lower binding activity to membrane TNFR and approximately 1400-fold 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 a 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 TNFa of Tyr^{1 15} 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 mammalian species by making the same amino acid substitutions in the analogous position of the respective species.

[042] Although muteins 1 , 2 and 4, as well as other TNFα muteins, were previously known and characterized with respect to binding the multivalent membrane receptor, it was previously unknown whether these TNFα muteins would bind to the monovalent soluble TNF receptors. As disclosed herein, the TNFα muteins bind with an affinity sufficient to block the binding of soluble TNF receptor to TNFα (see Example 2). These results indicate that TNFα muteins can be an effective binding partner for neutralizing soluble TNF receptor in a biological fluid.

[043] It is understood that TNFα muteins additional to the specific muteins exemplified herein can be used in methods of the invention. TNFα from various mammalian species show a high degree of amino acid identity (see Figure IA and IB, conserved sequence SEQ ID NO: 1 ; Van Ostade et al., supra, 1994). As described by Van Ostade et al. (supra , 1994), a conserved TNFα amino acid sequence was identified across 1 1 mammalian species. The conserved amino acid residues are conserved across all 1 1 shown species or have only a single species showing variation at that position (see Figure IA and Van Ostade et al., supra, 1994). Thus, in one embodiment, the invention provides a TNFα mutein comprising the conserved sequence referenced as SEQ ID NO: 1.

[044] One skilled in the art can readily determine additional TNFα muteins suitable for use in methods of the invention. As discussed above, TNFα muteins having relatively high affinity for TNF receptors and decreased signaling through membrane receptors, for example, decreased cytotoxicity or in vivo toxicity, relative to native TNFα are particularly useful in methods of the invention. One skilled in the art can readily determine additional suitable TNFα muteins based on methods well known to those skilled in the art. Methods for introducing amino acid substitutions into a sequence are well known to those skilled in the art (Ausubel et al., Current Protocols in Molecular Biology (Supplement 56), John Wiley & Sons, New York (2001); Sambrook and Russel, Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Press, Cold Spring Harbor (2001); U.S. Patent Nos. 5,264,563 and 5,523,388). Generation of TNFα muteins has been previously described (Van Ostade et al., supra, 1994; Van Ostade diύ., supra, 1991; Zhang eta\.,supra, 1992; Yamagishi et al., supra, 1990). Furthermore, one skilled in the art can readily determine the binding and cytotoxicity and/or in vivo toxicity of candidate muteins to ascertain the suitability for use in a method of the invention (Van Ostade et al., supra, 1994; Van Ostade et al., supra, 1991 ; Zhang et al., supra, 1992; Yamagishi et al., supra, 1990).

[045] TNFα muteins of particular interest for use in methods of the present invention, in addition to having relatively high affinity for TNF receptors and reduced signaling through membrane receptors, for example, reduced cytotoxicity or in vivo toxicity, are those having amino acid substitutions in three regions of TNFα, region 1, amino acids 29-36, region 2, amino acids 84-91, and region 3, amino acids 143-149 (numbering as shown in Figure IA). Muteins 1, 2 and 4 are exemplary of muteins having single amino acid substitutions in these regions. Region 1 corresponds to amino acids 29-36, residues LNRRANAL (SEQ ID NO:20) of human TNFα. Region 2 corresponds to amino acids 84-91, residues AVSYQTKV (SEQ ID NO:21) of human TNFα. Region 3 corresponds to amino acids 143-149, residues DFAESGQ (SEQ ID NO:22) of human TNFα. 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 screening for binding activity and cytotoxic activity and/or in vivo toxicity as disclosed herein (see also Van Ostade et al., supra, 1991 ; Zhang et al, supra, 1992; Yamagishi et al., supra, 1990). Methods for introducing amino acid substitutions at a particular amino acid residue or region 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; U.S. Patent 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 screened for binding activity and/or signaling activity, for example, cytotoxic activity or in vivo toxicity, to soluble and/or membrane bound TNF receptor. This would only require the generation of approximately 420 mutants (19 single amino acid substitutions at each of 22 positions in regions 1, 2 and 3), a number which can be readily generated and screened by well known methods. Those having 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 TNFα mutein useful in methods of the invention.

[046] The invention additionally provides a TNFα, mutein having the consensus sequence of SEQ ID NO:9 (Figure 1 C). In one embodiment, a TNFo; mutein comprises the consensus sequence SEQ ID NO:9, wherein Xl is an amino acid selected from Leu and VaI; wherein X2 is a 2 or 3 amino acid peptide having GIn or Arg at position 1 , Asn, Ala or Thr at position 2, and Ser, Leu, Pro or absent at position 3, for example, selected from GlnAsnSer, ArgAlaLeu, ArgThrPro, GlnAlaSer, and GlnThr; wherein X3 is an amino acid selected from Asp and Asn; wherein X4 is a 5 amino acid peptide having His, Pro, Leu, lie or VaI at position 1 , GIn, GIu, Ser, Asn or Lys at position 2, VaI, Ala or Ser at position 3, GIu or Pro at position 4, and GIu or GIy at position 5, for example, selected from HisGlnValGluGlu (SEQ ID NO:23), HisGlnAlaGluGlu (SEQ ID NO:24), ProGlnValGluGly (SEQ ID NO:25), ProGluAlaGluGly (SEQ ID NO:26), LeuSerAlaProGly (SEQ ID NO:27), IleSerAlaProGly (SEQ ID NO:28), ProGlnAlaGluGly (SEQ ID NO:29), IleAsnSerProGly (SEQ ID NO:30), and ValLysAlaGluGly (SEQ ID NO:31); wherein X5 is an amino acid selected from GIu, GIn and Arg; wherein X6 is a 4 amino acid peptide having Leu, GIy, Trp or GIn at position 1, Ser, Asp or Asn at position 2, GIn, Arg, Ser or GIy at position 3, and Arg or Tyr at position 4, for example, selected from LeuSerGlnArg (SEQ ID NO:32), LeuSerArgArg (SEQ ID NO:33), GlyAspSerTyr (SEQ ID NO:34), LeuSerGlyArg (SEQ ID NO:35), TφAspSerTyr (SEQ ID NO:36), GlnSerGlyTyr (SEQ ID NO:37), and LeuAsnArgArg (SEQ ID NO:38); wherein X7 is an amino acid selected from Leu, Met, and Lys; wherein X8 is a two amino acid peptide having Met or VaI at position 1 and Asp, Lys, GIu or GIn at position 2, for example, selected from MetAsp, MetLys, VaIGIu, ValLys, and VaIGIn; wherein X9 is an amino acid selected from Lys, Thr, GIu, and Arg; wherein XlO is an amino acid selected from VaI, Lys, and He; wherein XI l is a 2 amino acid peptide having Ala, Ser, Thr or Leu at position 1 and Asp or GIu at position 2, for example, selected from AlaAsp, SerAsp, ThrAsp, LeuAsp, AIaGIu, and SerGlu; wherein X 12 is an amino acid selected from Lys, Ser, Thr, and Arg; wherein 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, VaI, Tyr, Pro or Thr at position 2, VaI, Pro, His or Asn at position 3, Leu or VaI at position 4, and Leu, Phe or absent at position 5, for example, selected from AspValValLeu (SEQ ID NO:39), AspTyrValLeu (SEQ ID NO:40), SerTyrValLeu (SEQ ID NO:41), ProProProVal (SEQ ID NO:42), SerThrHisValLeu (SEQ ID NO:43), SerThrProLeuPhe (SEQ ID NO:44), SerThrHisValLeu (SEQ ID NO:45), and SerThrAsnValPhe (SEQ ID NO:46); wherein Xl 5 is an amino acid selected from VaI and He; wherein X16 is an amino acid selected from Phe, He, and Leu; wherein X17 is an amino acid selected from He and VaI; wherein X18 is a 2 amino acid peptide having GIn or Pro at position 1 and GIu, Asn, Thr or Ser at position 2, for example, selected from GInGIu, ProAsn, GlnThr, and ProSer; wherein Xl 9 is an amino acid selected from Leu and He; wherein X20 is a 3 amino acid peptide having Pro, His or GIn at position 1, Lys, Arg or Thr at position 2, and Asp or GIu at position 3, for example, selected from ProLysAsp, HisArgGlu, GlnArgGlu, and HisThrGlu; wherein X21 is an amino acid selected from GIy, GIu, 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 GIy and Asp; wherein X25 is an amino acid selected from GIn, Leu, and Arg; wherein X26 is an amino acid selected from Ala and Thr; wherein X27 is an amino acid selected from VaI and He; wherein X28 is an amino acid selected from Leu, GIn, and Arg; wherein X29 is an amino acid selected from Lys, GIu, 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 VaI and He (see Figure IA; Van Ostade et al., supra, 1994). Such a consensus TNFα mutein is expected to exhibit binding activity for TNF receptor, and such activity can be readily determined by those skilled in the art using well known methods, as disclosed herein.

[047] In addition to the variant positions described above, it is understood that a TNFα mutein can additionally include variant amino acids in the conserved sequence referenced as SEQ ID NO: 1. As shown in Figure IA and as discussed above, the conserved TNFα sequence includes certain positions where one of the shown mammalian species differs from the other ten. For example, the conserved amino acid at position 2, Arg, is Leu in dog (Figure IA). Thus, a TNFα mutein can include a substitution of Leu at position 2 with the remainder of the conserved sequence referenced as SEQ ID NO:1. Similarly, substitutions of other "conserved" positions, where at least one of the species has an amino acid substitution relative to the conserved sequence, are included as TNFα muteins. For example, a TNFo; mutein can have the corresponding substitution of mutein 1, that is, Arg³¹Pro and substitution in the conserved sequence in the variable positions, as described above represented by X, and/or substitution in a conserved position that varies in a single species. Furthermore, a TNFα mutein can include conservative amino acid substitutions relative to the conserved sequence or the sequence of a particular species of TNFa. Such TNFα muteins can be readily recognized by one skilled in the art based on the desired characteristics of a TNFα mutein, as disclosed herein.

[048] Additionally, any of the TNFα muteins disclosed herein can be modified to include an N-terminal deletion. As discussed in Van Ostade (supra, 1994), short deletions at the N-terminus of TNFα retained activity, whereas deletion of the N-terminal 17 amino acids resulted in a loss of activity. Therefore, it is understood that a TNFα mutein of the invention also includes TNFα muteins having N-terminal deletions that retain activity. Such TNFα muteins can include, for example, an N-terminal deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids. Furthermore, one skilled in the art can readily determine whether further N-terminal deletions can be incorporated into a TNFα mutein by making the deletion mutations and screening for desired characteristics, as disclosed herein. [049] The invention provides a variety of TNFα muteins, as disclosed herein. Generally, a particularly useful TNFa mutein of the invention has about 2-fold, about 3-fold, about 4- fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 1 1 -fold, about 12-fold, about 13-fold, about 14-fold, about 15-fold, about 16-fold, about 17- fold, about 18-fold, about 19- fold, about20-fold, about 25-fold, about 30-fold, about 50- fold, about 100-fold, or even higher fold reduced binding affinity for TNF receptors, particularly membrane bound TNF receptors, relative to native/wild type TNFα. Such reduced binding affinity can be, but is not necessarily, exhibited toward sTNFR. Also, a particularly useful TNFα mutein of the invention has about 10-fold, about 50-fold, about 100-fold, about 150- fold, about 200-fold, about 300-fold, about 500-fold, about 1000-fold, about 2000-fold, about 3000-fold, about 4000-fold, about 5000-fold, about 6000-fold, about 7000-fold, about 8000-fold, about 9000-fold, about 10,000-fold, about 20,000-fold, about 30,000-fold, about 50,000-fold, or even higher fold reduced signaling through the membrane receptors, for example, reduced cytoxicity or in vivo toxicity, relative to native/wild type TNFα. It is understood that a TNFα mutein can have reduced binding affinity and/or reduced cytoxicity, as discussed above and disclosed herein.

[050] The invention also provides dimeric forms of TNFα and TNFα muteins, which can be generated to further reduce the binding to, and signaling through, membrane TNFR. Native TNFα exists as a homotrimer of three 17 kDa subunits, as do the TNFα muteins exemplified herein. Other TNFα muteins produced using the methods described herein also can exist as trimers. The trimeric structure of TNFα or TNFα muteins allows for a contribution by avidity when binding to the membrane TNFR, which itself is multivalent by virtue of its high copy number on the cell surface. The dimeric form of TNFα has considerably lower affinity than trimeric TNFα for membrane TNFR due to a decreased contribution for avidity. Binding of TNFα or TNFα muteins to the monovalent soluble TNFR involves only two of the three monomers present in the TNFα or TNFα mutein trimer. While the TNFα or TNFα mutein trimer can bind to three monovalent soluble TNFR molecules, the binding to each is an independent event which is governed solely by affinity and which does not allow for a contribution by avidity. Therefore, the strength of binding by a dimeric form of TNFα or TNFα mutein to soluble TNFR is undiminished relative to the trimeric form of TNFα or TNFα muteins. In contrast, however, the binding of a dimeric form of TNFα or TNFα mutein to the membrane TNFR is reduced appreciably. Therefore, a dimeric form of a TNFα mutein can be used advantageously to preferentially bind to soluble TNFR over membrane TNFR.

[051] One method to generate a dimeric form of a TNFα or TNFα mutein is to produce a fusion protein which covalently links a TNFα or TNFα mutein monomer to an antibody heavy chain constant region. A particularly useful method to generate a dimeric TNFα is to fuse a TNFa to each of the two heavy chain constant regions involved in forming the dimeric Fc portion of an antibody. As these heavy chain constant region monomers assemble to form the stable Fc structure, they will cause the associated TNFα or TNFa mutein molecules to dimerize as well. In one embodiment, a fusion protein is produced in which the amino terminus of the TNFα mutein is fused to the carboxy-terminus of an intact heavy chain constant region. TNFα has been fused at its amino terminus in a variety of fusion proteins without a significant loss of biological activity. Such a heavy chain-TNFα mutein fusion protein can be assembled with intact antibody light chains to form a molecule that would neutralize soluble TNFR, have a significant half-life in vivo, and possess limited immunogenicity in a mammal from whom the antibody and TNFα sequences are derived. In another embodiment, the heavy chain constant region is truncated amino-terminal of the hinge region, thereby providing two sites to which a TNFα or TNFα mutein monomer can be fused at its carboxy terminus. Fusion proteins involving the carboxy terminus 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 TNFα (Leu¹⁵⁷) forms an ion pair with Lys" in an adjacent monomer, thus stabilizing trimer formation (Eck and Sprang, J. Biol. Chem. 264(29): 17595-17605 (1989), which is incorporated herein by reference). If the formation of a peptide bond that utilizes the carboxyl group of Leu¹⁵⁷ leads to diminished binding of the fusion protein for soluble TNFR, one or more amino acids can be inserted into the junction between the amino terminus of the heavy chain constant region and the carboxy terminus of the TNFα mutein, the amino acids having R-groups that contain carboxyl functionalities (for example, Asp or GIu). In yet another embodiment, other dimeric plasma proteins, including either homodimers or heterodimers, can be fused to the amino or carboxy termini of a TNFα mutein as described above.

[052] Alternatively, a TNFα monomer can be cross-linked to 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, Bioconjugate Techniques, Academic Press, San Diego (1996).

[053] In another embodiment, additional mutations can be introduced into the TNFo; or TNFα mutein portion of a dimeric fusion protein, such mutations being designed to reduce the ability of the dimerized TNFα or TNFα mutein to associate with a monomer of wild type TNFa. Association of dimeric TNFα or TNFα mutein with a monomer of wild type TNFα would restore the trimeric structure and potentially increase the ability of the fusion protein to bind to membrane TNFR and, thus, contribute to toxicity. The introduction of mutations at residues which normally form ion pairs that contribute to the assembly of trimeric TNFα or TNFα muteins (for example, Lys⁹⁸ and GIu^{1 16} or Lys" and Leu¹⁵⁷ , Eck and Sprang, supra, 1989) would reduce or eliminate the ability of such muteins to associate with wild type TNFα monomers. As discussed above, fusion of TNFα or TNFα muteins at the carboxy terminal Leu¹⁵⁷ to 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 Lys" in an adjacent subunit.

[054] The invention provides a method for reducing the levels of immune system inhibitors such as soluble TNF receptors in the circulation of a host mammal. The method can be used to potentiate an immune response, particularly in a mammal having a pathological condition for which the immune response can ameliorate a sign or symptom associated with the pathological condition.

[055] In one embodiment, the invention provides a method of neutralizing soluble tumor necrosis factor receptor (TNFR) in a mammal by administering an effective amount of a TNFα mutein having specific binding activity for a soluble TNFR , whereby binding of the TNFα mutein to the soluble TNFR neutralizes the soluble TNFR. The method can utilize a TNFα mutein, as disclosed herein, including a dimeric form of a TNFα mutein. [056] As used herein, "neutralizing," when referring to soluble TNFR, means that the amount of soluble TNFR available for binding to TNFα has been reduced. Thus, the effective concentration of soluble TNFR as it relates to TNFα binding is reduced, thereby effectively increasing the concentration of TNFα available for binding to membrane TNFR. This allows the available TNFα to bind to membrane TNFR and, for example, mediate its immune system stimulatory activity. TNFα muteins exhibiting preferential binding to soluble TNFR over membrane TNFR, as disclosed herein, are particularly useful for neutralizing soluble TNFR.

[057] In another embodiment, the invention provides a method of stimulating an immune response in a mammal having a pathological condition by administering an effective amount of a TNFα mutein having specific binding activity for a soluble tumor necrosis factor receptor (TNFR), whereby binding of the TNFα mutein to a soluble TNFR stimulates an immune response.

[058] In a particular embodiment of a method of the invention, the TNFα mutein can have specific binding activity for a single type of soluble TNFR, for example sTNFRI or sTNFRII . Alternatively, the TNFα mutein can have specific binding activity for more than one type of soluble TNFR, for example, both sTNFRI and sTNFRII

[059] The present invention further relates to the use of various mixtures of binding partners. One mixture can be composed of multiple binding partners that selectively bind to a single targeted immune system inhibitor. Another mixture can be composed of multiple binding partners, each of which selectively binds to different targeted immune system inhibitors. Alternatively, the mixture can be composed of multiple binding partners that selectively bind to different targeted immune system inhibitors. For example, the mixture can contain more than one TNFα mutein. Furthermore, the mutiple TNFα muteins can specifically bind to a single type of soluble TNF receptor or can bind to more than one type of TNF receptor, for example, sTNFRI and sTNFRII.

[060] In another embodiment of a method of the invention, a plurality of TNFα muteins can be administered. In a particular embodiment, the plurality of TNFα muteins can have specific binding activity for a single type of soluble TNFR, for example, sTNFRI or sTNFRII. Alternatively, the plurality of TNFα muteins can have specific binding activity for more than one type of soluble TNFR, that is, sTNFRI and sTNFRII.

[061] As used herein, "functionally active binding sites" of a binding partner refer to sites that are capable of binding to one or more targeted immune system inhibitors.

[062] Methods for producing the various binding partners useful in the present invention are well known to those skilled in the art. Such methods include, for example, recombinant DNA and synthetic techniques, or a combination thereof. Binding partners such as TNFα muteins can be expressed in prokaryotic or eukaroytic cells, for example, mammalian, insect, yeast, and the like. If desired, codons can be changed to reflect any codon bias in a host species used for expression.

[063] If desirable, the entire process can be repeated. Those skilled in the art can readily determine the benefits of repeated treatment by monitoring the clinical status of the patient, and correlating that status with the concentration(s) of the targeted immune system inhibitor(s) such as soluble TNFα receptor in circulation prior to, during, and after treatment.

[064] In a specific embodiment, the immune system inhibitor to be targeted is sTNFRI (Seckinger et al., J. Biol. Chem. 264: 11966-11973 (1989); Gatanaga et al., Proc. Natl. Acad. Sci. USA 87:8781-8784 (1990)), a naturally occurring inhibitor of the pluripotent immune system stimulator, TNF. sTNFRI is produced by proteolytic cleavage, which liberates the extra-cellular domain of the membrane tumor necrosis factor receptor type I from its transmembrane and intracellular domains (Schall et al., Cell 61:361-370 (1990); Himmler et al., DNA and Cell Biol. 9:705-715 (1990)). sTNFRI retains the ability to bind to TNF with high affinity and, thus, to inhibit the binding of TNF to the membrane receptor on cell surfaces.

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

[066] In tumor bearing hosts, over-production of sTNFRI may profoundly affect the course of disease, considering the critical role of TNF in a variety of anti-tumor immune responses (reviewed in, Beutler and Cerami, Ann. Rev. Immunol. 7:625-655 (1989)). TNF directly induces tumor cell death by binding to the type I membrane-associated TNF receptor. Moreover, the death of vascular endothelial cells is induced by TNF binding, destroying the circulatory network serving the tumor and further contributing to tumor cell death. Critical roles for TNF in natural killer cell- and cytotoxic T lymphocyte-mediated cytolysis also have been documented. Inhibition of any or all of these effector mechanisms by sTNFRI has the potential to dramatically enhance tumor survival.

[067] That sTNFRI promotes tumor survival, and that its removal enhances anti-tumor immunity, has been demonstrated. In an experimental mouse tumor model, sTNFRI production was found to protect transformed cells in litro from the cytotoxic effects of TNF, and from 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 enhance their tumorigenicity and persistence in vivo (Selinsky and Howell. Cell. Immunol. 200:81-87 (2000)). Moreover, removal of circulating sTNFRI has been found to provide clinical benefit to cancer patients, as demonstrated by human trials of Ultrapheresis as discussed above (Lentz, supra, 1989, 1997, 1999). These observations affirm the importance of this molecule in tumor survival and suggest the development of methods for more specific removal of sTNFRI as promising new avenues for cancer immunotherapy.

[068] For use as a therapeutic agent, the TFNα mutein can be formulated with a pharmaceutically acceptable carrier to produce a pharmaceutical composition, which can be administered to the individual, which can be a human or other mammal. A pharmaceutically acceptable carrier can be, for example, water, sodium phosphate buffer, phosphate buffered saline, normal saline or Ringer's solution or other physiologically buffered saline, or other solvent or vehicle such as a glycol, glycerol, an oil such as olive oil or an injectable organic ester. The therapeutic compositions of the invention can also contain a carrier or excipient, many of which are known to one of ordinary skill in the art. Excipients that can be used include buffers, for example, citrate buffer, phosphate buffer, acetate buffer, and bicarbonate buffer; amino acids; urea; alcohols; ascorbic acid; glutathione; phospholipids; proteins, for example, serum albumin; ethylenediamine tetraacetic acid (EDTA); sodium chloride or other salts; liposomes; mannitol, sorbitol, glycerol, glucose, sucrose, dextrans; calcium or magnesium, and the like. The agents of the invention can be formulated in various ways, according to the corresponding route of administration. For example, liquid solutions can be made for ingestion or injection; gels or powders can be made for ingestion, inhalation, or topical application. Methods for making such formulations are well known and can be found in, for example, "Remington's Pharmaceutical Sciences," 18th ed., Mack Publishing Company, Easton PA (1990).

[069] A pharmaceutical composition containing a TNFα mutein can be administered to an individual by various routes, including by intravenous, subcutaneous, intramuscular, intrathecal or intraperitoneal injection; orally, as an aerosol spray; or by intubation. If desired, the TNFα mutein can be incorporated into a liposome, a non-liposome lipid complex, or other polymer matrix, which further can have incorporated therein, for example, a second drug useful for treating the individual. Liposomes, which consist of phospholipids or other lipids, are nontoxic, physiologically acceptable and metabolizable carriers that are relatively simple to make and administer (Gregoriadis, Liposome Technology, Vol. 1 (CRC Press, Boca Raton FL, 1984), which is incorporated herein by reference). The skilled artisan can readily determine an appropriate route and method of administration. It is understood that other therapies suitable for treating a particular pathological condition can also be administered during a course of administration with a TNFo; mutein. The other therapy can be co-administered with the TNFa mutein, separately administered, or administered alternately or on different regimens, as desired.

[070] The following examples are put forth to provide those of ordinary skill in the art with a complete disclosure and description of how the compositions and/or methods claimed herein are made and evaluated, and are intended to be purely exemplary of the invention and are not intended to limit the scope of what the inventors regard as the invention. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. The present invention is more particularly described in the following examples which are intended as illustrative only because numerous modifications and variations therein will be apparent to those skilled in the art.

EXAMPLE 1

Production, Purification, and Characterization of the Immune System Inhibitor,

Human sTNFRI

[071] The sTNFRI used in the present studies was produced recombinantly either in E. coli (R&D Systems; Minneapolis MN) or in eukaryotic cell culture essentially as described (see U.S. Patent No. 6,379,708, which is incorporated herein by reference). The construction of the eukaryotic expression plasmid, the methods for transforming and selecting cultured cells, and for assaying the production of sTNFRI by the transformed cells have been described (Selinsky et al., supra, 1998).

[072] sTNFRI was detected and quantified in the present studies by capture ELISA (Selinsky et al., supra). In addition, the biological activity of recombinant sTNFRI, that is, its ability to bind TNF, was confirmed by ELISA. Assay plates were coated with human TNF a (Chemicon; Temecula CA), blocked with bovine serum albumin, and sTNFRI, contained in culture supernatants as described above, was added. Bound sTNFRI was detected through the sequential addition of biotinylatedgoat anti-human sTNFRI, alkaline phosphatase- conjugated streptavidin, and pnitrophenylphosphate.

EXAMPLE 2 Production, Purification, and Characterization of TNFα Muteins

[073] Briefly, TNFα muteins 1, 2, 3 and 4 were produced by expression of the respective cDNAs in E. coli. Genes encoding 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 frame to that encoding the ompA leader to permit export of the recombinant polypeptides to the periplasm. Synthetic fragments were cloned into a pUC19 derivative immediately downstream of the lac Z promoter, and the resulting recombinant plasmids were introduced into E. coll. Recombinant bacteria were cultured to late-log, induced with isopropyl-B-D-thiogalactopyranoside (IPTG) for three hours, and harvested by centrifugation. Periplasmic fractions were prepared and tested by ELISA using polyclonal goat anti-human TNFα capture antibodies. After the addition of the diluted periplasms, bound TNFo: and TNFα muteins 1, 2, 3 and 4 were detected by sequential addition of biotinylated polyclonal goat anti-human TNFo; streptavidinalkaline phosphatase, and para-nitrophenyl phosphate (pNPP). TNFα and each of the TNFα muteins was detectable in the respective periplasms, though the level of TNFa mutein 3 only slightly exceeded the detection limit of the assay (Figure 2).

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

[075] The TNFα muteins were tested for their ability to bind to sTNFRI. Wells of a microtiter plate were coated with TNFα, blocked, and incubated with sTNFRI either in the presence or absence of TNFα and TNFα muteins 1, 2 and 4. As shown in Figure 3, TNFα muteins 1 , 2 and 4 each bind to sTNFRI and inhibit its binding to TNFα.

[076] Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this pertains.

[077] Although the invention has been described with reference to the presently preferred embodiments, 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

What is claimed is:

1. A dimeric tumor necrosis factor a (TNFα) mutein.

2. The dimeric TNFa mutein of claim 1, wherein the TNFα mutein comprises the conserved sequence referenced as SEQ ID NO:1.

3. The dimeric TNFα a mutein of claim 1, wherein the TNFα mutein has the consensus sequence SEQ ID NO:9, wherein Xi is an amino acid selected from Leu and VaI;

wherein X2 is a 2 or 3 amino acid peptide selected from GlnAsnSer, ArgAlaLeu, ArgThrPro, GlnAlaSer, and GlnThr; wherein X3 is an amino acid selected from Asp and Asn; whereinX4 is a 5 amino acid peptide selected fiυm HisGInValGluGlu, HisGlnAlaGluGlu, ProGlnValGluGly, ProGluAlaGluGly, LeuSerAlaProGly, IleSerAlaProGly, ProGlnAlaGluGly, IleAsnSerProGly, and ValLysAlaGluGly; wherein X5 is an amino acid selected from GIu, GIn and Arg;

wherein X6 is a 4 amino acid peptide selected from LeuSerGlnArg, LeuSerArgArg, GlyAspSerTyr, LeuSerGlyArg, TφAspSerTyr, GlnSerGlyTyr, and LeuAsnArgArg;

wherein X7 is an amino acid selected from Leu, Met, and Lys;

wherein X8 is a two amino acid peptide selected from MetAsp, MetLys, VaIGIu, ValLys, and VaIGIn; wherein X9 is an amino acid selected from Lys, Thr, GIu, and Arg; wherein XlO is an amino acid selected from VaI, Lys, and He; wherein Xl 1 is a 2 amino acid peptide selected from AlaAsp, SerAsp, Thr Asp, LeuAsp, AIaGIu, and SerGlu; wherein X 12 is an amino acid selected from Lys, Ser, Thr, and Arg; wherein X 13 is an amino acid selected from GIn and His; wherein X 14 is a 4 or 5 amino acid peptide selected from AspValValLeu, AspTyrValLeu, SerTyrValLeu, ProProProVal, SerThrHisValLeu, SerThrProLeuPhe, SerThrHisValLeu, and SerThrAsnValPhe;

wherein X 15 is an amino acid selected from VaI and He; wherein X16 is an amino acid selected from Phe, He, and Leu; wherein Xl 7 is an amino acid selected from He and VaI;

wherein Xl 8 is a 2 amino acid peptide selected from GInGIu, ProAsn, GlnThr, and ProSer;

wherein Xl 9 is an amino acid selected from Leu and He;

wherein X20 is a 3 amino acid peptide selected from ProLysAsp, HisArgGlu, GlnArgGlu, and HisThrGlu; wherein X21 is an amino acid selected from GIy, GIu, 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 GIy and Asp; wherein X25 is an amino acid selected from GIn, Leu, and Arg; wherein X26 is an amino acid selected from Ala and Thr; wherein X27 is an amino acid selected from VaI and He; wherein X28 is an amino acid selected from Leu, GIn, and Arg;

wherein X29 is an amino acid selected from Lys, GIu, 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 VaI and He.

4. The dimeric TNFα mutein of claim 1 , wherein the TNFa, mutein has an amino acid substitution in a region of TNFα selected from region 1 amino acids 2936, 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 from another species.

5. The dimeric TNFα mutein of claim 1 , wherein the TNFα mutein 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. The dimeric TNFα mutein of claim 1, wherein the TNFα mutein is selected from mutein 1 (SEQ ID NO:3), mutein 2 (SEQ ID NO:4), and mutein 4 (SEQ ID NO:6).

7. The dimeric TNFα mutein of claim 1, wherein the TNFα mutein is derived from a species selected from human, dog, cat, horse, sheep, goat, pig, cow, rabbit and rat.

8. The dimeric TNFα mutein of claim 1, wherein said dimeric TNFα mutein is a fusion with a Fc.

9. A method of neutralizing soluble tumor necrosis factor receptor (TNFR) in a mammal, comprising administering an effective amount of a tumor necrosis factor a (TNFα) mutein having specific binding activity for a soluble TNFR, whereby binding of said TNFα mutein to said soluble TNFR neutralizes said soluble TNFR.

10. The method of claim 9, wherein the TNFα mutein comprises the conserved sequence referenced as SEQ ID NO:1.

11. The method of claim 9, wherein the TNFα mutein has the consensus sequence SEQ ID NO:9,

wherein Xl is an amino acid selected from Leu and VaI;

wherein X2 is a 2 or 3 amino acid peptide selected from GlnAsnSer, ArgAlaLeu, ArgThrPro, GlnAlaSer, and GlnThr; wherein X3 is an amino acid selected from Asp and Asn;

wherein X4 is a 5 amino acid peptide selected from HisGlnValGluGlu, HisGlnAlaGluGlu, ProGlnValGluGly, ProGluAlaGluGly, LeuSerAlaProGly, IleSerAlaProGly, ProGlnAlaGluGly, IleAsnSerProGly, and ValLysAlaGluGly;

wherein X5 is an amino acid selected from GIu, GIn and Arg;

wherein X7 is an amino acid selected from Leu, Met, and Lys;

wherein X8 is a two amino acid peptide selected from MetAsp, MetLys, VaIGIu, ValLys, and VaIGIn;

wherein X9 is an amino acid selected from Lys, Thr, GIu, and Arg;

wherein XlO is an amino acid selected from VaI, Lys, and He;

wherein XI l is a 2 amino acid peptide selected from AlaAsp, SerAsp, ThrAsp, LeuAsp, AIaGIu, and SerGlu;

wherein Xl 2 is an amino acid selected from Lys, Ser, Thr, and Arg;

wherein Xl 3 is an amino acid selected from GIn and His;

wherein X 14 is a 4 or 5 amino acid peptide selected from AspValValLeu, AspTyrValLeu, SerTyrValLeu, ProProProVal, SerThrHisValLeu, SerThrProLeuPhe, SerThrHisValLeu, and SerThrAsnValPhe;

wherein Xl 5 is an amino acid selected from VaI and He;

wherein Xl 6 is an amino acid selected from Phe, He, and Leu;

wherein X17 is an amino acid selected from He and VaI; wherein X 18 is a 2 amino acid peptide selected from GinGlu, ProAsn, GlnThr, and ProSer;

wherein X19 is an amino acid selected from Leu and He;

wherein X20 is a 3 amino acid peptide selected from ProLysAsp, HisArgGlu, GlnArgGlu, and HisThrGlu;

wherein X21 is an amino acid selected from GIy, GIu, 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 GIy and Asp;

wherein X25 is an amino acid selected from GIn, Leu, and Arg;

wherein X26 is an amino acid selected from Ala and Thr;

wherein X27 is an amino acid selected from VaI and He;

wherein X28 is an amino acid selected from Leu, GIn, and Arg;

wherein X29 is an amino acid selected from Lys, GIu, 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 VaI and He.

12. The method of claim 9, wherein the TNFα mutein 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 TNFa(SEQ ID NO:2) or the analogous position of TNFα from another species.

13. The method of claim 9, wherein the TNFa mutein 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).

14. The method of claim 9, wherein the TNFa mutein is selected from mutein 1 (SEQ ID NO:3), mutein 2 (SEQ ID NO:4), and mutein 4 (SEQ ID NO:6).

15. The method of claim 9, wherein said TNFα mutein is dimeric.

16. The method of claim 15, wherein said dimeric TNFα mutein is a fusion with a Fc.

17. The method of claim 9, wherein said TNFα mutein has specific binding activity for a single type of soluble TNFR.

18. The method of claim 17, wherein said soluble TNFR is sTNFRI.

19. The method of claim 17, wherein said soluble TNFR is sTNFRII.

20. The method of claim 9, wherein the TNFα mutein has specific binding activity for more than one type of soluble TNFR.

21. The method of claim 20, wherein said TNFα mutein has specific binding activity for sTNFRI and sTNFRII.

22. The method of claim 9, wherein said TNFα mutein is produced recombinantly.

23. The method of claim 9, wherein a plurality of TNFα muteins is administered.

24. The method of claim 23, wherein said plurality of TNFα muteins has specific binding activity for a single type of soluble TNFR.

25. The method of claim 23, wherein said soluble TNFR is sTNFRI.

26. The method of claim 23, wherein soluble TNFR is sTNFRII.

27. The method of claim 9, wherein said plurality of TNFα muteins has specific binding activity for more than one type of soluble TNFR.

28. The method of claim 27, wherein said plurality of TNFα muteins has specific binding activity for sTNFRI and sTNFRII.

29. The method of claim 9, wherein the mammal is human.

30. The method of claim 9, wherein the mammal is non-human.

31. A method of stimulating an immune response in a mammal having a pathological condition, comprising administering an effective amount of a tumor necrosis factor a (TNFα) mutein having specific binding activity for a soluble tumor necrosis factor receptor (TNFR), whereby binding of said TNFα mutein to a soluble TNFR stimulates an immune response.

32. The method of claim 31, wherein the TNFα mutein comprises the conserved sequence referenced as SEQ ID NO:1.

33. The method of claim 31 , wherein the TNFα mutein has the consensus sequence SEQ ID NO:9,

wherein Xl is an amino acid selected from Leu and VaI;

wherein X2 is a 2 or 3 amino acid peptide selected from GlnAsnSer, ArgAlaLeu, ArgThrPro, GlnAlaSer, and GinThr;

wherein X3 is an amino acid selected from Asp and Asn;

wherein X5 is an amino acid selected from GIu, GIn and Arg;

wherein X6 is a 4 amino acid peptide selected from LeuSerGlnArg, LeuSerArgArg, GlyAspSerTyr, LeuSerGlyArg, TrpAspSerTyr, GlnSerGlyTyr, and LeuAsnArgArg;

wherein X7 is an amino acid selected from Leu, Met, and Lys;

wherein X9 is an amino acid selected from Lys, Thr, GIu, and Arg;

wherein XlO is an amino acid selected from VaI, Lys, and He;

wherein X 12 is an amino acid selected from Lys, Ser, Thr, and Arg;

wherein Xl 3 is an amino acid selected from GIn and His; wherein X 14 is a 4 or 5 amino acid peptide selected from AspValValLeu, AspTyrValLeu, SerTyrValLeu, ProProProVal, SerThrHisValLeu, SerThrProLeuPhe, SerThrHis ValLeu, and SerThrAsnValPhe;

wherein Xl 5 is an amino acid selected from VaI and He;

wherein X16 is an amino acid selected from Phe, He, and Leu;

wherein Xl 7 is an amino acid selected from lie and VaI;

wherein X18 is a 2 amino acid peptide selected from GInGIu, ProAsn, GlnThr, and ProSer;

wherein Xl 9 is an amino acid selected from Leu and He;

wherein X21 is an amino acid selected from GIy, GIu, 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 GIy and Asp;

wherein X25 is an amino acid selected from GIn, Leu, and Arg;

wherein X26 is an amino acid selected from Ala and Thr;

wherein X27 is an amino acid selected from VaI and He;

wherein X28 is an amino acid selected from Leu, GIn, and Arg;

wherein X29 is an amino acid selected from Lys, GIu, 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 VaI and He.

34. The method of claim 31 , wherein the TNFα mutein 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 from another species.

35. The method of claim 31, wherein the TNFα mutein 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).

36. The method of claim 31, wherein the TNFa mutein is selected from mutein 1 (SEQ ID NO:3), mutein 2 (SEQ ID NO:4), and mutein 4 (SEQ ID NO:6).

37. The method of claim 31, wherein said TNFα mutein is dimeric.

38. The method of claim 37, wherein said dimeric TNFα mutein is a fusion with a Fc.

39. The method of claim 31, wherein said TNFα mutein has specific binding activity for a single type of soluble TNFR.

40. The method of claim 39, wherein said soluble TNFR is sTNFRI.

41. The method of claim 39, wherein said soluble TNFR is sTNFRII.

42. The method of claim 31, wherein the TNFα, mutein has specific binding activity for more than one type of soluble TNFR.

43. The method of claim 42, wherein said TNFα mutein has specific binding activity for sTNFRI and sTNFRII.

44. The method of claim 31, wherein said TNFα mutein is produced recombinantly.

45. The method of claim 31, wherein a plurality of TNFα, muteins is administered.

46. The method of claim 45, wherein said plurality of TNFα, muteins has specific binding activity for a single type of soluble TNFR.

47. The method of claim 45, wherein said soluble TNFR is sTNFRI.

48. The method of claim 45, wherein soluble TNFR is sTNFRII.

49. The method of claim 31, wherein said plurality of TNFα muteins has specific binding activity for more than one type of soluble TNFR.

50. The method of claim 49, wherein said plurality of TNFα muteins has specific binding activity for sTNFRI and sTNFRII.

51. The method of claim 31 , wherein the mammal is human.

52. The method of claim 31 , wherein the mammal is non-human.

53. 53. A dimeric tumor necrosis factor a (TNFo;).