IMMOBILIZED TUMOR NECROSIS FACTOR-ALPHA MUTEINS FOR ENHANCING IMMUNE RESPONSE IN MAMMALS
20170143889 ยท 2017-05-25
Inventors
Cpc classification
A61P31/00
HUMAN NECESSITIES
A61M1/38
HUMAN NECESSITIES
A61M1/3615
HUMAN NECESSITIES
A61P35/00
HUMAN NECESSITIES
International classification
A61M1/36
HUMAN NECESSITIES
A61M1/38
HUMAN NECESSITIES
Abstract
An extracorporeal system for enhancing an immune response in a mammal to facilitate the elimination of a chronic pathology. The system includes an absorbent matrix capable of removing an 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 partner(s) such as TNF- muteins capable of binding to and, thus, depleting the targeted immune system inhibitor(s) from the biological fluids. The absorbent matrix may comprise an inert, biocompatible substrate joined covalently to a binding partner, such as a TNF- mutein, capable of specifically binding to a targeted immune system inhibitor such as soluble TNF receptor.
Claims
1. An absorbent matrix comprising: a plurality of tumor necrosis factor-alpha (TNF-) immobilized on an extracorporeal substrate; wherein the plurality of TNF- muteins have at least one amino acid substitution relative to an unsubstituted native TNF-; and wherein each of the plurality of TNF- muteins immobilized on the extracorporeal substrate have at least one binding site capable of selectively binding to a soluble TNF receptor with an affinity sufficient to deplete the soluble TNF receptor from a biological fluid.
2. The absorbent matrix of claim 1, wherein the plurality of TNF- muteins are selected from the group consisting of 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), or combinations thereof.
3. The absorbent matrix of claim 1, wherein the plurality of TNF- muteins comprise a conserved mutein sequence (SEQ ID NO:1).
4. The absorbent matrix of claim 1, wherein the plurality of TNF- muteins comprise the consensus mutein sequence (SEQ ID NO:9), wherein X.sub.1 is an amino acid selected from Leu and Val; wherein X.sub.2 is a 2 or 3 amino acid peptide selected from GlnAsnSer, ArgAlaLeu, ArgThrPro, GlnAlaSer, and GlnThr; wherein X.sub.3 is an amino acid selected from Asp and Asn; wherein X.sub.4 is a 5 amino acid peptide selected from HisGlnValGluGlu, HisGlnAlaGluGlu, ProGlnValGluGly, ProGluAlaGluGly, LeuSerAlaProGly, IleSerAlaProGly, ProGlnAlaGluGly, IleAsnSerProGly, and ValLysAlaGluGly; wherein X.sub.5 is an amino acid selected from Glu, Gln and Arg; wherein X.sub.6 is a 4 amino acid peptide selected from LeuSerGlnArg, LeuSerArgArg, GlyAspSerTyr, LeuSerGlyArg, TrpAspSerTyr, GinSerGlyTyr, and LeuAsnArgArg; wherein X.sub.7 is an amino acid selected from Leu, Met, and Lys; wherein X.sub.g is a two amino acid peptide selected from MetAsp, MetLys, ValGlu, ValLys, and ValGln; wherein X.sub.9 is an amino acid selected from Lys, Thr, Glu, and Arg; wherein X.sub.10 is an amino acid selected from Val, Lys, and Ile; wherein X.sub.11 is a 2 amino acid peptide selected from AlaAsp, SerAsp, ThrAsp, LeuAsp, AlaGlu, and SerGlu; wherein X.sub.12 is an amino acid selected from Lys, Ser, Thr, and Arg; wherein X.sub.13 is an amino acid selected from Gln and His; wherein X.sub.14 is a 4 or 5 amino acid peptide selected from AspValValLeu, AspTyrValLeu, SerTyrValLeu, ProProProVal, SerThrHisValLeu, SerThrProLeuPhe, SerThrHisValLeu, and SerThrAsnValPhe; wherein X.sub.15 is an amino acid selected from Val and Ile; wherein X.sub.16 is an amino acid selected from Phe, Ile, and Leu; wherein X.sub.17 is an amino acid selected from Ile and Val; wherein X.sub.18 is a 2 amino acid peptide selected from GlnGlu, ProAsn, GlnThr, and ProSer; wherein X.sub.19 is an amino acid selected from Leu and Ile; wherein X.sub.20 is a 3 amino acid peptide selected from ProLysAsp, HisArgGlu, GlnArgGlu, and HisThrGlu; wherein X.sub.21 is an amino acid selected from Gly, Glu, Gln, and Trp or is absent; wherein X.sub.22 is an amino acid selected from Leu, Pro, and Ala; wherein X.sub.23 is an amino acid selected from Leu and Gln; wherein X.sub.24 is an amino acid selected from Gly and Asp; wherein X.sub.25 is an amino acid selected from Gln, Leu, and Arg; wherein X.sub.26 is an amino acid selected from Ala and Thr; wherein X.sub.27 is an amino acid selected from Val and Ile; wherein X.sub.28 is an amino acid selected from Leu, Gin, and Arg; wherein X.sub.29 is an amino acid selected from Lys, Glu, Ala, Asn, and Asp; wherein X.sub.30 is an amino acid selected from Phe, Ile, Leu and Tyr; and wherein X.sub.31 is an amino acid selected from Val and Ile.
5. The absorbent matrix of claim 1, wherein the plurality of TNF- muteins have an amino acid substitution in a region of TNF- selected from region 1 amino acids 29-36, region 2 amino acids 84-91, and region 3 amino acids 143-149 of human TNF- (SEQ ID NO:2) or an analogous position of TNF- from another species.
6. The absorbent matrix of claim 1, wherein the extracorporeal substrate is a biocompatible solid support.
7. The absorbent matrix of claim 6, wherein the biocompatible solid support is in the form of a bead.
8. The absorbent matrix of claim 7, wherein the bead is a macroporous bead.
9. The absorbent matrix of claim 8, wherein the macroporous bead is chosen from agarose, cross-linked agarose, cellulose, controlled pore glass, polyacrylamide, azlactone, polymethacrylate and polystyrene.
10. The aborbent matrix of claim 1, wherein the unsubstituted native TNF- is a human TNF-.
11. The absorbent matrix of claim 1, wherein the soluble TNF receptor is a soluble tumor necrosis factor receptor Type I (sTNFRI) or a soluble tumor necrosis factor receptor Type II (sTNFRII).
12. An extracorpeal system for reducing the amount of a targeted immune system inhibitor in blood of a donor mammal, the extracorpeal system comprising: an apheresis device in fluid communication with a blood source from the donor mammal that separates the whole blood component into a conduit containing a cellular component and a conduit containing an acellular component or a fraction of an acellular component, the acellular component or the fraction of the acellular component containing the targeted immune system inhibitor comprising a soluble TNF receptor; an absorbent matrix in fluid communication with the apheresis device by the conduit containing the acellular component or the fraction of the acellular component, the absorbent matrix having a plurality of TNF- muteins immobilized on an inert medium; the plurality of TNF- muteins having at least one amino acid substitution relative to an unsubstituted native TNF-; and each of the plurality of TNF- muteins immobilized on the extracorporeal substrate having at least one binding site capable of selectively binding to a soluble TNF receptor with an affinity sufficient to deplete the soluble TNF receptor from the acellular component to produce an altered acellular component or an altered fraction of the acellular component contained in a conduit exiting the absorbent matrix; and a volumetric pump in fluid communication with the apheresis device that provides a pressure differential to the conduit containing the cellular component and the conduit containing the altered acellular component or the altered fraction of the acellular component to combine the cellular component with the altered acellular component or the altered fraction of the acellular component to produce an altered whole blood source adapted to be administered to a recipient mammal.
13. The extracorpeal system of claim 12, wherein the conduit containing the cellular component is a first conduit and the conduit containing the acellular component or the fraction of the acellular component is a second conduit.
14. The extracorpeal system of claim 12, wherein the donor mammal of the blood source is the recipient mammal.
15. The extracorpeal system of claim 12, further comprising a positive displacement blood pump that removes the blood source from the donor mammal.
16. The extracorporeal system of claim 12, wherein the inert medium is selected from the group consisting of a hollow fiber, a macroporous bead, a cellulose-based fiber, a synthetic fiber, a flat membrane, a pleated membrane, and a silica-based particle.
17. The extracorporeal system of claim 12, wherein the blood source is whole blood.
18. The extracorporeal system of claim 12, wherein the plurality of TNF- muteins are selected from the group consisting of 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), or combinations thereof.
19. The extracorporeal system of claim 12, wherein the soluble TNF receptor is a soluble tumor necrosis factor receptor Type I (sTNFRI) or a soluble tumor necrosis factor receptor Type II (sTNFRII).
20. The extracorporeal system of claim 12, wherein the plurality of TNF- muteins have an amino acid substitution in a region of TNF- selected from region 1 amino acids 29-36, region 2 amino acids 84-91, and region 3 amino acids 143-149 of human TNF- (SEQ ID NO:2) or an analogous position of TNF- from another species.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0023]
[0024]
[0025]
[0026]
[0027]
[0028]
DETAILED DESCRIPTION OF THE DRAWINGS
[0029] The present invention provides methods to reduce the levels of 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.
[0030] The methods of the present invention generally are accomplished by: (a) obtaining a biological fluid from a mammal having a pathological condition; (b) contacting the biological fluid with a TNF mutein binding partner capable of selectively binding to a targeted immune system inhibitor such as soluble TNF receptor to produce an altered biological fluid having a reduced amount of the targeted immune system inhibitor; and, thereafter (c) administering the altered biological fluid to the mammal.
[0031] 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 and , interleukin-1, interleukin-2, interleukin-4, interleukin-5, interleukin-6, interleukin-8, interleukin-12, interferon-.gamma., interferon-7; and the chemokines RANTES, macrophage inflammatory proteins 1- and 1- and macrophage chemotactic and activating factor, as discussed above.
[0032] 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-y and tumor necrosis factors and . 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, soluble receptors for tumor necrosis factor and . As used herein, the term targeted immune system inhibitor refers to that inhibitor, or collection of inhibitors, which is to be removed from the biological fluid by a method of the invention, for example, sTNFRI and/or sTNFRII.
[0033] As used herein, the term soluble TNF receptor refers to a soluble form of a receptor for TNF and TNF. 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 TNF and TNF 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 remove one or both types of TNF receptor depending on whether the TNF mutein or plurality of muteins used in the method binds to one or both types of receptors.
[0034] 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.
[0035] 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, supra, 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.
[0036] 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. If desired, the biological fluid can be processed or fractionated, for example, to obtain an acellular component. As it relates to the present invention, 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 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 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 therapeutic benefit. As an initial guideline, one plasma volume (approximately 3-5 liters in an adult human) can be removed and, thereafter, depleted of the targeted immune system inhibitor according to the present methods.
[0037] 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.
[0038] 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 or 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. Those 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 TNF mutein.
[0039] As used herein, the term TNF mutein refers to a TNF variant having one or more amino acid substitutions relative to a parent sequence and retaining specific binding activity for a TNF receptor. Generally, the muteins of the present invention have a single amino acid substitution relative to a parent sequence. Exemplary TNF muteins include the human TNF muteins designated muteins 1, 2, 3, 4, 5 and 6 (see
[0040] The present invention provides compositions and method for 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, also referred to herein as binding partners, can be attached to a solid support to allow the removal of an immune system inhibitor from a biological fluid.
[0041] 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. Generally, even when a ligand such as a binding partner is covalently bound to a solid support, a certain percentage of the bound ligand will leach from the support, for example, via chemical reactions that break down the covalent linkage or protease activity present in a biological fluid. In such a case, the ligand will leach into the biological fluid being processed and, thus, be returned to the patient. Therefore, 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, even if some of the ligand leaches into the processed biological fluid, the ligand would still exhibit low biological activity with respect to membrane receptor signaling when reintroduced into the patient.
[0042] Yet another useful characteristic of a binding partner is a lack of indirect toxicity, for example, immunogenicity. As discussed above, it is common for a bound ligand to leach from a matrix, resulting in the ligand being present in the processed biological fluid. Because the biological fluid is returned 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 which the process 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 TNF mutein can be used as the binding partner, which is expected to have low immunogenicity given the homology to the endogenous TNF. Similarly, muteins derived from other mammalian species can be used in the respective species.
[0043] As disclosed herein, TNF muteins are particularly useful binding partners in methods of the invention. A number of TNF 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 TNF muteins shown in
[0044] There are several advantages to using TNF muteins as binding partners in the present invention. Although TNF muteins can display lower binding activity for TNF receptors, some TNF muteins bind only 5- to 17-fold less effectively than native TNF. Such a binding affinity, albeit reduced relative to native TNF, can still be an effective binding partner in the present invention (see Example 3). Another advantage of using TNF muteins is that some exhibit decreased signaling through membrane receptors, for example, decreased cytotoxic activity or in vivo toxicity, relative to native TNF. 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 in view of the potential leaching of the ligand from a matrix and introduction of low levels into a patient when an altered biological fluid is returned to the patient.
[0045] An additional advantage of using TNF muteins is that they have a native structure. Because the muteins are highly homologous to the native TNF 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 TNF, except for possibly the mutant amino acid residue. Such a native folding means that the TNF muteins should have little or no immunogenicity in the respective mammalian species.
[0046] As disclosed herein, particularly useful muteins are human muteins 1, 2, 3, 4, 5 and 6 (
[0047] Although muteins 1, 2 and 4, as well as other TNF muteins, were previously known and characterized with respect to binding the multivalent membrane receptor, it was previously unknown whether these TNF muteins would bind to the monovalent soluble TNF receptors. As disclosed herein, the TNF muteins bind with an affinity sufficient to deplete soluble TNF receptor from plasma (see Example 3). These results indicate that TNF muteins can be an effective binding partner for depleting soluble TNF receptor from a biological fluid.
[0048] It is understood that TNF muteins additional to the specific muteins exemplified herein can be used in methods of the invention. TNF from various mammalian species show a high degree of amino acid identity (see
[0049] One skilled in the art can readily determine additional muteins suitable for use in methods of the invention. As discussed above, TNF 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 TNF are particularly useful in methods of the invention. One skilled in the art can readily determine additional suitable TNF 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. Pat. Nos. 5,264,563 and 5,523,388). Generation of TNF muteins 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). 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).
[0050] 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 TNF, region 1, amino acids 29-36, region 2, amino acids 84-91, and region 3, amino acids 143-149 (numbering as shown in
[0051] The invention additionally provides a TNF mutein having the consensus sequence of SEQ ID NO:9 (
[0052] In addition to the variant positions described above, it is understood that a TNF mutein can additionally include variant amino acids in the conserved sequence referenced as SEQ ID NO:1. As shown in
[0053] Additionally, any of the TNF 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 TNF retained activity, whereas deletion of the N-terminal 17 amino acids resulted in a loss of activity. Therefore, it is understood that a TNF mutein of the invention also includes TNF muteins having N-terminal deletions that retain activity. Such TNF 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 TNF mutein by making the deletion mutations and screening for desired characteristics, as disclosed herein.
[0054] The invention provides a variety of TNF muteins, as disclosed herein. Generally, a particularly useful TNF 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 11-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, about 20-fold, about 25-fold, about 30-fold, or even higher fold reduced binding affinity for TNF receptors, particularly membrane bound TNF receptors, relative to native/wild type TNF. Such reduced binding affinity can be, but is not necessarily, exhibited toward sTNFR. Also, a particularly useful TNF 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 TNF. It is understood that a TNF mutein can have reduced binding affinity and/or reduced cytoxicity, as discussed above and disclosed herein.
[0055] The invention provides a conjugate comprising a tumor necrosis factor (TNF) mutein attached to a substrate. In one embodiment, the TNF mutein of the conjugate comprises the conserved sequence referenced as SEQ ID NO:1.
[0056] In another embodiment, the invention provides a conjugate where the TNF mutein has the consensus sequence SEQ ID NO:9, wherein X.sub.1 is an amino acid selected from Leu and Val; wherein X.sub.2 is a 2 or 3 amino acid peptide selected from GlnAsnSer, ArgAlaLeu, ArgThrPro, GlnAlaSer, and GlnThr; wherein X.sub.3 is an amino acid selected from Asp and Asn; wherein X.sub.4 is a 5 amino acid peptide selected from HisGlnValGluGlu, HisGlnAlaGluGlu, ProGInValGluGly, ProGluAlaGluGly, LeuSerAlaProGly, IleSerAlaProGly, ProGlnAlaGluGly, IleAsnSerProGly, and ValLysAlaGluGly; wherein X.sub.5 is an amino acid selected from Glu, Gln and Arg; wherein X.sub.6 is a 4 amino acid peptide selected from LeuSerGlnArg, LeuSerArgArg, GlyAspSerTyr, LeuSerGlyArg, TrpAspSerTyr, GlnSerGlyTyr, and LeuAsnArgArg; wherein X.sub.7 is an amino acid selected from Leu, Met, and Lys; wherein X.sub.8 is a two amino acid peptide selected from MetAsp, MetLys, ValGlu, ValLys, and ValGln; wherein X.sub.9 is an amino acid selected from Lys, Thr, Glu, and Arg; wherein X.sub.10 is an amino acid selected from Val, Lys, and Ile; wherein X.sub.11 is a 2 amino acid peptide selected from AlaAsp, SerAsp, ThrAsp, LeuAsp, AlaGlu, and SerGlu; wherein X.sub.12 is an amino acid selected from Lys, Ser, Thr, and Arg; wherein X.sub.13 is an amino acid selected from Gln and His; wherein X.sub.14 is a 4 or 5 amino acid peptide selected from AspValValLeu, AspTyrValLeu, SerTyrValLeu, ProProProVal, SerThrHisValLeu, SerThrProLeuPhe, SerThrHisValLeu, and SerThrAsnValPhe; wherein X.sub.15 is an amino acid selected from Val and Ile; wherein X.sub.16 is an amino acid selected from Phe, Ile, and Leu; wherein X.sub.17 is an amino acid selected from Ile and Val; wherein X.sub.18 is a 2 amino acid peptide selected from GlnGlu, ProAsn, GlnThr, and ProSer; wherein X.sub.19 is an amino acid selected from Leu and Ile; wherein X.sub.20 is a 3 amino acid peptide selected from ProLysAsp, HisArgGlu, GlnArgGlu, and HisThrGlu; wherein X.sub.21 is an amino acid selected from Gly, Glu, Gln, and Trp or is absent; wherein X.sub.22 is an amino acid selected from Leu, Pro, and Ala; wherein X.sub.23 is an amino acid selected from Leu and Gln; wherein X.sub.24 is an amino acid selected from Gly and Asp; wherein X.sub.25 is an amino acid selected from Gln, Leu, and Arg; wherein X.sub.26 is an amino acid selected from Ala and Thr; wherein X.sub.27 is an amino acid selected from Val and Ile; wherein X.sub.28 is an amino acid selected from Leu, Gln, and Arg; wherein X.sub.29 is an amino acid selected from Lys, Glu, Ala, Asn, and Asp; wherein X.sub.30 is an amino acid selected from Phe, Ile, Leu and Tyr; and wherein X.sub.31 is an amino acid selected from Val and Ile.
[0057] In yet another embodiment, the invention provides a conjugate where the TNF mutein has an amino acid substitution in a region of TNF selected from region 1 amino acids 29-36, region 2 amino acids 84-91 and region 3 amino acids 143-149 of human TNF (SEQ ID NO:2) or the analogous position of TNF from another species.
[0058] In still another embodiment, the invention provides a conjugate where the TNF 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). In a particular embodiment, the invention provides a conjugate where the TNF mutein is selected from mutein 1 (SEQ ID NO:3), mutein 2 (SEQ ID NO:4), and mutein 4 (SEQ ID NO:6). The TNF mutein of the conjugate can be derived from a species selected, for example, from human, dog, cat, horse, sheep, goat, pig, cow, rabbit and rat.
[0059] The invention additionally provides a method of stimulating an immune response in a mammal having a pathological condition. The method can include the steps of obtaining a biological fluid from the mammal; contacting the biological fluid with a tumor necrosis factor (TNF) mutein having specific binding activity for a soluble tumor necrosis factor receptor (TNFR); removing the TNF mutein bound to the 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 can be, for example, blood, plasma, serum or lymphatic fluid, including whole blood. In one embodiment, a method using whole blood as the biological fluid can 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 or the fraction of the acellular component contains a soluble TNFR. The method can additionally include the step of combining the cellular component with the altered acellular component or altered fraction of the acellular component to produce altered whole blood, which is administered to the mammal as the altered biological fluid.
[0060] In a particular embodiment of a method of the invention, the TNF mutein can have specific binding activity for a single type of soluble TNFR, for example sTNFRI or sTNFRII. Alternatively, the TNF mutein can have specific binding activity for more than one type of soluble TNFR, for example, both sTNFRI and sTNFRII.
[0061] 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 TNF mutein. Furthermore, the multiple TNF 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.
[0062] In another embodiment of a method of the invention, the biological fluid can be contacted with a plurality of TNF muteins. In a particular embodiment, the plurality of TNF muteins can have specific binding activity for a single type of soluble TNFR, for example, sTNFRI or sTNFRII. Alternatively, the plurality of TNF muteins can have specific binding activity for more than one type of soluble TNFR, that is, sTNFRI and sTNFRII.
[0063] For certain embodiments in which it is desirable to increase the molecular weight of the binding partner/immune system inhibitor complex, 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.
[0064] 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.
[0065] 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 TNF 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.
[0066] In one embodiment of the present methods, the binding partner such as a TNF mutein is attached to an inert medium to form an absorbent matrix (
[0067] The absorbent matrix thus produced can be contacted with a biological fluid, or a fraction thereof, through the use of an extracorporeal circuit. The development and use of extracorporal, absorbent matrices has been extensively reviewed (see Kessler, Blood Purification 11:150-157 (1993)).
[0068] In another embodiment, herein referred to as the stirred reactor (
[0069] In a final step of the present methods, the treated or altered biological fluid, having a reduced amount of targeted immune system inhibitor such as soluble TNF receptor, is returned to the patient receiving treatment along with untreated fractions of the biological fluid, if any such fractions were produced during the treatment. The altered biological fluid can be administered to the mammal by any means known to those skilled in the art, including, for example, by infusion directly into the circulatory system. The altered biological fluid can be administered immediately after contact with the binding partner in a contemporaneous, extracorporeal circuit. In this circuit, the biological fluid is (a) collected, (b) separated into cellular and acellular components, if desired, (c) exposed to the binding partner, and if needed, separated from the binding partner bound to the targeted immune system inhibitor, (d) combined with the cellular component, if needed, and (e) readministered to the patient as altered biological fluid. Alternatively, the administration of the altered biological fluid can be delayed under appropriate storage conditions readily determined by those skilled in the art.
[0070] 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 TNF receptor in circulation prior to, during, and after treatment.
[0071] The present invention further provides an apparatus for reducing the amount of a targeted immune system inhibitor such as soluble TNF receptor in a biological fluid. The apparatus is composed of: (a) a means for separating the biological fluid into a cellular component and an acellular component or fraction thereof; (b) an absorbent matrix having attached thereto a TNF mutein or a stirred reactor as described above to produce an altered acellular component or fraction thereof; and (c) a means for combining the cellular fraction with the altered acellular component or fraction thereof. The apparatus is particularly useful for whole blood as the biological fluid in which the cellular component is separated either from whole plasma or a fraction thereof.
[0072] The means for initially fractionating the biological fluid into the cellular component and the acellular component, or a fraction thereof, and for recombining the cellular component with the acellular component, or fraction thereof, after treatment are known to those skilled in the art (see Apheresis: Principles and Practice, supra).
[0073] 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.
[0074] 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.
[0075] 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.
[0076] 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 vitro 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). 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.
[0077] The following examples are intended to illustrate but not limit the invention.
EXAMPLE 1
[0078] Production, Purification, and Characterization of the Immune System Inhibitor, Human sTNFRI
[0079] The sTNFRI used in the present studies was produced recombinantly either in E. coli (R&D Systems; Minneapolis, Minn.) or in eukaryotic cell culture essentially as described (see U.S. Pat. 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).
[0080] 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 (Chemicon; Temecula, Calif.), 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 biotinylated-goat anti-human sTNFRI, alkaline phosphatase-conjugated streptavidin, and p-nitrophenylphosphate.
EXAMPLE 2
[0081] Production, Purification, and Characterization of TNF Muteins
[0082] Briefly, TNF muteins 1, 2, 3 and 4 were produced by expression of the respective cDNAs in E. coli. Genes encoding TNF and TNF 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. coli. Recombinant bacteria were cultured to late-log, induced with isopropyl--D-thiogalactopyranoside (IPTG) for three hours, and harvested by centrifugation. Periplasmic fractions were prepared and tested by ELISA using polyclonal goat anti-human TNF capture antibodies. After the addition of the diluted periplasms, bound TNF and TNF muteins 1, 2, 3 and 4 were detected by sequential addition of biotinylated polyclonal goat anti-human TNF, streptavidin-alkaline phosphatase, and para-nitrophenyl phosphate (pNPP). TNF and each of the TNF muteins were detectable in the respective periplasms, though the level of TNF mutein 3 only slightly exceeded the detection limit of the assay (
[0083] The TNF and TNF 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. Mol. Biol. 211:493-501 (1990)). The TNF and TNF 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 TNF or the muteins and a 34 kDa band, which was confirmed by Western blotting to be dimerized TNF mutein.
[0084] The TNF muteins were tested for their ability to bind to sTNFRI. Wells of a microtiter plate were coated with TNF, blocked, and incubated with sTNFRI either in the presence or absence of the inhibitors, TNF and TNF muteins 1, 2 and 4. As shown in
EXAMPLE 3
[0085] Depletion of the Immune System Inhibitor, sTNFRI, From Human Plasma Using TNF Mutein Absorbent Matrices
[0086] The TNF mutein absorbent matrices were produced and tested for their ability to deplete sTNFRI from human plasma. Briefly, purified TNF muteins 1, 2 and 4 each was conjugated to cyanogen bromide (CNBr) Sepharose 4B at a density of 0.5 mg per ml of beads, and the remaining CNBr groups were quenched with ethanolamine. The resulting matrices were packed in individual column housings and washed extensively with phosphate buffered saline prior to use.
[0087] Normal human plasma was spiked (33% v/v) with culture supernatant containing recombinant human sTNFRI (see Example 1) to 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 no immobilized protein and quenched with ethanolamine, was included to control for non-specific depletion. One ml fractions 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, wells were coated with polyclonal goat anti-sTNFRI, and then were blocked with 2% BSA. Plasma samples were diluted 1:2. added to the wells, and sTNFRI therein was captured. Biotinylated polyclonal goat anti-sTNFRI was added, followed by streptavidin-alkaline phosphatase, and p-nitrophenylphosphate. Relative absorbance at 405 nm was used to estimate the depletion.
[0088] As shown in
[0089] 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.