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[0002] 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.
[0003] 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.
[0004] 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.,
[0005] 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, W. P.,
[0006] 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 α and β, interferon-γ, interleukin-1, interleukin-2, interleukin-4, interleukin-6, and interleukin-7.
[0007] 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.
[0008] Third, particular immune system inhibitors exert their effects by binding to receptors on host cells and signalling a decrease in their production of immune system stimulators. Examples include, but are not limited to, interferon-β, which decreases the production of two key proinflammatory mediators, tumor necrosis factor-α and interleukin-1 (Coclet-Ninin et al.,
[0009] 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-β inhibits a variety of immune cells, and significantly limits inflammation and cell-mediated immune responses (Reviewed in, Letterio and Roberts,
[0010] 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, M. K.,
[0011] 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-β dominates over the production of immunostimulatory mediators (i.e., interleukin-2, interferon-γ, and/or tumor necrosis factor-β) (Reviewed in, Lucey, et al.,
[0012] In addition 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.,
[0013] Direct evidence that the removal of immune system inhibitors provides clinical benefit derives from the evaluation of Ultrapheresis, a promising experimental cancer therapy (Lentz, M. R.,
[0014] Ultrapheresis is in direct contrast to more traditional approaches which have endeavored to boost immunity through the addition of immune system stimulators. Pre-eminent among these has been the infusion of supraphysiological levels of TNF (Sidhu and Bollon,
[0015] Although Ultrapheresis provides advantages over traditional therapeutic approaches, there are certain drawbacks 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.
[0016] Because of the beneficial effects associated with the removal of immune system inhibitors, there exists a need for methods which can be used to specifically deplete those inhibitors from circulation. Such methods ideally should be specific and not remove other circulatory components, and they should not result in any significant loss of circulatory volume. The present invention satisfies these needs and provides related advantages as well.
[0017] The present invention provides a method for stimulating immune responses in a mammal through the depletion of immune system inhibitors present in the circulation of said mammal. The depletion of immune system inhibitors can be effected by removing biological fluids from said mammal and contacting these biological fluids with a binding partner capable of selectively binding to the targeted immune system inhibitor.
[0018] Binding partners useful in these methods can be antibodies, both polyclonal or monoclonal antibodies, or fractions thereof, having specificity for a targeted immune system inhibitor. Additionally, binding partners to which the immune system inhibitor naturally binds may be used. Synthetic peptides created to attach specifically to targeted immune system inhibitors also are useful as binding partners in the present methods. Moreover, mixtures of binding partners having specificity for multiple immune system inhibitors may be used.
[0019] In a particularly useful embodiment, the binding partner is immobilized previously on a solid support to create an “absorbent matrix” (
[0020] In another embodiment, the binding partner can be mixed with the biological fluid in a “stirred reactor” (
[0021] The present invention also provides apparatus incorporating either the absorbent matrix or the stirred reactor.
[0022]
[0023]
[0024]
[0025] The present invention provides novel methods to reduce the levels of immune system inhibitors in the circulation of a host mammal, thereby, potentiating an immune response capable of resolving a pathological condition. By enhancing the magnitude of the host's immune response, the invention avoids the problems associated with the repeated administration of chemotherapeutic agents which often have undesirable side effects (e.g., chemotherapeutic agents used in treating cancer).
[0026] 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 binding partner capable of selectively binding to a targeted immune system inhibitor 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 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.
[0027] 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-β, 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 α and β. Immune system inhibitors produced by microorganisms are also potential targets including, for example, complement inhibitors, and homologues of interleukin-10, soluble receptors for interleukin-1, interferons α, β, and γ, and tumor necrosis factors α and β. 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 the present method.
[0028] As used herein, the term “mammal” can be a human or a non-human animal, such as dog, cat, horse, cattle, pig, or sheep for example. The term “patient” is used synonymously with the term “mammal” in describing the invention.
[0029] 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). 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.
[0030] As it relates to the present invention, the term “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:
[0031] 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”.
[0032] 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 α or 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.
[0033] 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 different binding sites on a single targeted immune system inhibitor. Another mixture can be composed of multiple binding partners, each of which selectively binds to a single site on different targeted immune system inhibitors. Alternatively, the mixture can be composed of multiple binding partners that selectively bind to different binding sites on different targeted immune system inhibitors. The mixtures referred to above may include mixtures of antibodies or fractions thereof, mixtures of natural binding partners, mixtures of synthetic peptides, or mixtures of any combinations thereof.
[0034] For certain embodiments in which it would be 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.
[0035] Additionally, binding partners can be constructed as multifunctional antibodies according to methods known in the art.
[0036] For example, bifunctional antibodies having two functionally active binding sites per molecule or trifunctional antibodies having three functionally active binding sites per molecule can be made by known methods. As used herein, “functionally active binding sites” refer to sites that are capable of binding to one or more targeted immune system inhibitors. By way of illustration, a bifunctional antibody can be produced that has functionally active binding sites, each of which selectively binds to different targeted immune system inhibitors.
[0037] Methods for producing the various binding partners useful in the present invention are well known to those skilled in the art.
[0038] Such methods include, for example, serologic, hybridoma, recombinant DNA, and synthetic techniques, or a combination thereof.
[0039] In one embodiment of the present methods, the binding partner is attached to an inert medium to form an absorbent matrix (
[0040] 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 extracorporeal, absorbent matrices has been extensively reviewed. (See, Kessler, L.,
[0041] In another embodiment, herein referred to as the “stirred reactor” (
[0042] In the final step of the present methods, the treated or altered biological fluid, having a reduced amount of targeted immune system inhibitor, 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.
[0043] It may be desirable to repeat the entire process. 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) in circulation prior to, during, and after treatment.
[0044] The present invention further provides novel apparatus for reducing the amount of a targeted immune system inhibitor in a biological fluid. These apparatus are 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 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. These apparatus are particularly useful for whole blood as the biological fluid in which the cellular component is separated either from whole plasma or a fraction thereof.
[0045] 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:
[0046] In one specific embodiment, the immune system inhibitor to be targeted is sTNFRI (Seckinger, et al.,
[0047] 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.
[0048] 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,
[0049] 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.,
[0050] The following examples are intended to illustrate but not limit the invention.
[0051] The sTNFRI used in the present studies was produced recombinantly in cell culture. The construction of the eukaryotic expression plasmid, the methods for transforming cultured cells, and for assaying the production of sTNFRI by the transformed cells have been described (Selinsky, et al., supra). The sTNFRI expression plasmid was introduced into HeLa cells (American Type Culture Collection #CCL 2), and an sTNFRI-producing transfectant cell line was isolated by limiting dilution. This cloned cell line was cultured in a fluidized-bed reactor at 37° C. in RPMI-1640, supplemented with 2.5% (v/v) fetal bovine serum and penicillin/streptomycin, each at 100 micrograms per milliliter. sTNFRI secreted into the culture medium was purified by affinity chromatography on a TNF-Sepharose-4B affinity matrix essentially as described (Engelmann, et al.,
[0052] sTNFRI was detected and quantified in the present studies by capture ELISA (Selinsky, et al., supra). In addition, the biological activity of recombinant sTNFRI, i.e., its ability to bind TNF, was confirmed by ELISA. Assay plates were coated with human TNF-α (Chemicon), blocked with bovine serum albumin, and sTNFRI, purified from 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 ρ-nitrophenylphosphate.
[0053] Binding partners used in the present studies include an IgG fraction of goat anti-human sTNFRI antisera (R&D Systems, Cat.#AF425-PB) and a monoclonal antibody reactive with sTNFRI (Biosource International, Cat.#AHR3912). An additional monoclonal antibody, OT145 (Cat.#TCR1657), reactive with a human T cell receptor protein, was purchased from T Cell Diagnostics (now, Endogen) and was used as a control binding partner. Each of these respective binding partners was covalently conjugated to cyanogen bromide-activated Sepharose-4B (Pharmacia Biotech), a macroporous bead which facilitates the covalent attachment of proteins. Antibodies were conjugated at 1.0 milligram of protein per milliliter of swollen gel, and the matrices were washed extensively according to the manufacturer's specifications. Matrices were equilibrated in phosphate buffered saline prior to use.
[0054] Normal human plasma was spiked with purified sTNFRI to a final concentration of 10 nanograms per milliliter, a concentration comparable to those found in the circulation of cancer patients (Gadducci, et al., supra). One milliliter of the spiked plasma was mixed with 0.25 milliliter of the respective absorbent matrices at 0° C. and a plasma sample was removed at time=0. The samples were warmed rapidly to 37° C., and incubated with agitation for an additional 45 minutes. Plasma samples were removed for analysis at 15 minute intervals and, immediately after collection, were separated from the beads by centrifugation. Samples were analyzed by ELISA to quantify the levels of sTNFRI, and to permit the determination of the extent of depletion.
[0055]
[0056] 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.