WO2005007218A2

WO2005007218A2 - Ex vivo perfusion

Info

Publication number: WO2005007218A2
Application number: PCT/US2004/022249
Authority: WO
Inventors: Bruce E. Reidenberg; Ronald M. Burch
Original assignee: Euro-Celtique S.A.
Priority date: 2003-07-11
Filing date: 2004-07-09
Publication date: 2005-01-27
Also published as: WO2005007218A3; WO2005007218A8

Abstract

A method of removing a target molecule from blood by contacting the blood with an ex vivo perfusion device comprising binding partners for the target molecule is provided. The ex vivo perfusion device preferably comprises one or more immobilized synthebodies as binding partners. Also provided is a method for treating a patient suffering from symptoms associated with an adenoma or other endocrine tumor. The method comprises contacting the blood or a blood component of the patient with an ex vivo perfusion device to which one or more binding partners of a hormone secreted by the tumor are immobilized. The tumor can be, for example, an insulinoma or a parathyroid hormone (PTH) adenoma, and the hormone can be, for example, insulin, PTH, glucagon, VIP, and somatostatin. The binding partner can be, e.g., a synthebody or a part thereof.

Description

Ex Vivo Perfusion

This application claims priority to U.S. Serial No. 60/486,794, filed on July 11, 2003. This prior application is incorporated herein by reference.

FIELD OF THE INVENTION [001] The present invention relates to methods of reducing adverse effects in patients suffering from cancer. Specifically, this invention relates to the removal of elevated levels of peptide hormones produced by tumors in patients suffering from cancers such as adenoma by contacting the patient's blood or a component thereof e vivo with a binding partner such as, for example, an immunoglobulin, a variant of an immunoglobulin molecule, or a portion of such an immunoglobulin molecule.

BACKGROUND OF THE INVENTION [002] Certain adenoma tumors secrete super-physiological levels of normal peptide hormones. These super-physiological hormone levels cause metabolic havoc, and can be the bases for pathology of these types of adenomas. Generally, resection of the tumor resolves the problem. The difficulty in finding and/or removing these tumors, however, complicates that approach, and a patent may fall victim to the effects of elevated peptide hormone levels before the tumor can be located and removed.

[003] One example of a hormone secreted in excess by certain adenomas is human parathyroid hormone (PTH). PTH is secreted by the parathyroid as an 84 amino acid protein, and is synthesized in the body from a 90-amino acid precursor called ProPTH, which is itself an intermediary material synthesized from a protein which contains 115 amino acids, Pre- ProPTH. N- terminal fragments of PTH, particularly those consisting of amino acids 1-34 and 1-38, retain the full biological activity of the intact protein. PTH is involved in calcium and phosphorus homeostasis and control of bone growth and density, for example, by participating in regulating metabolic activities of bone and kidney and the intestinal absorption of calcium in warm blooded animals, such as humans. Importantly, PTH protects organisms from severe hypocalcemia. Thus, when the circulating concentration of ionized calcium is low, the secretion rate of PTH is stimulated.

[004] Normally, PTH is secreted in physiological dosages that vary according to the body's requirements at any specific time. Adenomas of the parathyroid gland, however, often cause overproduction of PTH. When PTH secretion is excessive and does not occur in its normal dosage-time related manner responsive to metabolic requirements, calcium levels become elevated in serum, and undue bone resorption and demineralization occurs, leading to hypocalcemia and toxic effects in soft tissues. Elevated serum calcium can result in various constitutional symptoms, the formation of kidney stones, and the loss of bone density in patients with hyperparathyroidism. The treatment of choice for hypeφarathyroidism has been surgical removal of any parathyroid adenomas, but these tumors can be difficult to locate surgically. An adenoma, the location of which is unknown, can peφetuate continual hormone secretion, causing substantial damage. PTH and its effects are described in The Role of Calcium in Biological Systems, Vol. II, Anghileri CRC Press, Boca Raton, Fla., 1981, Chapter II, pages 204-212.

[005] Insulin is another example of a constitutive molecule that can be excessively produced in patients suffering from certain adenomas. In normal physiological states, the pancreas produces insulin. While the majority of pancreatic cancers have moφhologic characteristics of cells of the exocrine pancreas, some pancreatic cancers are derived from the very cells secreting insulin, i.e., the beta cells. These tumors are referred to as insulinomas. Pancreatic insulinoma can cause excess levels of insulin and proinsulin, leading to hypoglycemia. Other types of gastro-enteropancreatic neoplasms associated with excessive excretion of hormones include glucagonoma, associated with oveφroduction of glucagon, leading to diabetes and diarrhea; the Verner-Morrison syndrome, caused by oveφroduction of vasoactive intestinal peptide (VIP) from pancreatic islet tumors and associated with severe secretory diarrhea; and somatostatinoma, secreting excessive levels of somatostatin that can lead to, e.g., gallbladder dysfunction and impaired glucose tolerance (Tomassetti et al., Ann Oncol 2001;12:S95-9). [006] Plasma perfusion techniques have been applied to remove selected toxic and harmful substances from blood in both specific and non-specific manners. This technique generally involves removing blood from a patient, separating the cellular components of the blood from the plasma component, and passing the plasma through a device which removes the undesirable substances. The cellular blood components and treated plasma are then returned, either together or separately, to the patient. Alternatively, the cellular components may be combined with replacement plasma, and the newly constituted blood can be infused into the patient. There are many types of continuous and intermittent blood processing methods, each of which provide different therapeutic effects and demand different processing criteria.

[007] In adsorbent-based plasma perfusion, the undesirable component is removed by contacting plasma with a binding partner that is specific for the undesirable component, and wherein the binding partner is attached to a solid support. The binding partner can be an antibody or antibody fragment directed against the undesirable component, in which case the technique is referred to as "immunoadsoφtion." For example, U.S. Patent No. 4,512,763 to Schneider discloses the use of an antibody as an immunoadsorbent to remove various components of a patient's blood, including immune complexes. U.S. Patent No. 6,287,516 to Matson et al. describes the use of an anti-TNF antibody to remove inflammatory mediators such as tumor necrosis factor (TNF). In addition, extracoφoreal ("outside the body") perfusion has been used as a treatment for AIDS and other immune deficiencies, wherein blood is passed through a solid support containing antibodies to remove defective interferon molecules. (U.S. Patent No. 4,824,432 to Skurkovich et al.).

[008] Binding partners other than antibodies can be utilized. For example, ex vivo adsoφtion techniques based on synthetic blood-group antigens attached to a solid support have been suggested to remove anti-A and anti-B antibodies, thereby overcoming blood- group incompatibility (PCT International Publication WO 00/74824 by Bensinger). Extracoφoreal perfusion has been used to reduce the blood levels of therapeutic agents such as radiolabeled and biotinylated anti-tumor antibodies, after a sufficient amount of the agent has bound to the tumor, by passing plasma through a perfusion device containing immobilized avidin (U.S. Patent No. 6,251,394 to Nilsson et al ). Ex vivo perfusion techniques using a catalytic molecule, instead of a binding partner, have also been disclosed. For example, U.S. Patent No. 6,004,768 to Navia et al. discloses an extracoφoreal perfusion device containing asparaginase to break down circulating asparagine in certain cancer chemotherapy regimens.

[009] Such protocols for abating a pathological response by ex vivo perfusion techniques all rely upon identifying a key or pivotal single cytokine or mediator target for inactivation or removal, the most widely accepted methods being binding with monoclonal antibodies ("MoAbs") or with specific antagonists ("SA").

[0010] There is a need for improved techniques to specifically target peptide hormones and other molecules for removal from the circulatory system. Such a system would be particularly useful in treating various tumor-related diseases or conditions involving the endocrine system such as, for example, those resulting from parathyroid hormone adenoma or pancreatic insulinoma.

SUMMARY OF THE INVENTION [0011] The present invention advantageously provides a method of treating a tumor- associated disease or condition such as, but not limited to, that caused by an adenoma such as parathyroid hormone adenoma or pancreatic insulinoma, and other diseases or conditions that result in unwanted hormones or elevated amounts of hormones in the blood.

[0012] The present invention also provides a method of removing a target molecule from blood or a blood component, which method comprises contacting ex vivo the blood or the blood component containing an undesirable level of the target molecule with an immobilized synthebody specific for the target molecule under conditions that result in the binding of the target molecule to the synthebody. In one embodiment, the target molecule is produced in a subject as a consequence of an illness, disease or condition. For example, the illness, disease or condition can be a tumor. Exemplary tumors include adenomas.

[0013] The target molecule may be constitutively present at a level lower than the undesirable level in blood of a normal subject, or may not be present in blood of a normal subject. In a preferred embodiment, the target molecule is a peptide. Target peptides useful in the present invention include insulin, parathyroid hormone (PTH), glucagon, somatostatin, and vasoactive intestinal peptide (VEP). For example, PTH may result from a PTH-secreting parathyroid adenoma, and insulin may result from an insulin-secreting pancreatic insulinoma.

[0014] In one embodiment, the contacting comprises perfusion. The perfusion may be carried out with an assisting pump. The synthebody is preferably, although not necessarily, immobilized on a solid support, and the solid support can be incoφorated in a hemofiltration device. In a particular embodiment, a second synthebody specific for a different target molecule is immobilized on the support. The solid support may comprise, for example, beads, plates, and/or hollow filters.

[0015] The synthebody can comprise a variant of an immunoglobulin variable domain, the immunoglobulin variable domain comprising: (i) at least one CDR region and (ii) framework regions flanking the CDR, wherein the variant comprises the CDR region having added or substituted therein at least one amino acid sequence which is heterologous to the CDR and the flanking framework regions, and the heterologous sequence is capable of specifically binding to the molecule. In preferred embodiments, the heterologous sequence comprises at least 10 sequential amino acid residues of SEQ ID NO: 3 or 6. The immunoglobulin is preferably an antibody or antibody fragment.

[0016] The invention also provides an apparatus comprising a hemofiltration device comprising a solid support having a synthebody binding partner of a peptide hormone immobilized thereron.

[0017] The invention also provides a method of treating an adverse effect caused by a parathyroid hormone adenoma in a patient in need of such treatment, the method comprising removing a parathyroid hormone produced by the adenoma from the blood of the patient by contacting ex vivo the blood or a blood component with an immobilized synthebody binding partner of the hormone. In one embodiment, the synthebody binding partner is specific for a parathyroid hormone comprising an amino acid sequence as set forth in SEQ ID NO:l or 2. IN another embodiment, the synthebody binding partner comprises at least 10 sequential amino acid residues of SEQ ID NO: 3. [0018] The invention also provides for a method of treating an adverse effect or a condition caused by a pancreatic insulinoma in a patient in need of such treatment, the method comprising removing insulin produced by the pancreatic insulinoma from the blood of the patient by contacting ex vivo the blood or a blood component with an immobilized synthebody binding partner of the insulin. In one embodiment, the synthebody binding partner is specific for an insulin sequence comprising at least one amino acid sequence as set forth in SEQ ID NO: 4 or 5. In another embodiment, the synthebody binding partner comprises at least 10 sequential amino acid residues of SEQ ID NO: 6. In still another embodiment, insulin is present, or is present at an elevated level, in the blood as a result of the presence of the pancreatic insulinoma, and the method can be used to treat such a condition.

[0019] The invention also provides for a method of treating hypeφarathyroidism in a patient in need of such treatment, the method comprising removing a parathyroid hormone from the blood of the patient by contacting ex vivo the blood or a blood component with an immobilized synthebody binding partner of the hormone. The hormone may comprise, e.g., the amino acid sequence of SEQ ID NOS: 1 or 2. In one embodiment, the binding partner comprises at least 10 sequential amino acid residues of SEQ ID NO: 3. This method may be used to treat hypeφarathyroidism, or a condition resulting therefrom, in a patient in need of such treatment. For example, the PTH may be present, or may be present at an elevated level, as a result of a parathyroid adenoma in the patient.

BRIEF DESCRIPTION OF DRAWINGS [0020] FIGURE 1. Graphical illustration of PCR knitting.

[0021] FIGURE 2. Exemplary design of a perfusion device according to the invention.

DETAILED DESCRIPTION [0022] The present invention provides a method for removing a specific target molecule from blood, or from a blood component such as plasma or serum. This method is useful for a variety of puφoses including, e.g., the elimination, or the reduction of an excess amount, of a peptide hormone from the blood or blood component. In one embodiment, the peptide hormone is secreted by a tumor tissue. The target molecule can be present in the blood or blood component, or is present in the blood or blood component in an elevated level compared to the level in a "normal" blood sample, due to the presence of a tumor in the subject or patient from whom the blood or blood component has been obtained.

[0023] The method of the present invention comprises contacting, in an ex vivo apparatus, specific target molecules present in the blood or blood component with antibody or antibody variant molecules such as "synthebodies" that specifically bind to such target molecules under conditions that allow binding to occur between the antibody or synthebody and target molecules. The method enables the removal of all or a portion of the amount of the target molecule without exposing a subject directly to the binding partners, e.g., synthebodies. Preferably, the target molecule is "specifically removed", meaning that all or a portion of the amount of target molecules removed from the blood or blood component without the removal of other peptides, hormones or other blood constituents.

[0024] The binding partner, e.g., the antibody or synthebody, is preferably immobilized on a solid support, which can be incoφorated in a hemofiltration device or other device known in the art. In one embodiment, a second binding partner, such as synthebody specific for a different target molecule, is also immobilized. Thus, the support can be used to remove two different target molecules.

[0025] The synthebody comprises a variant of an immunoglobulin variable domain, wherein the immunoglobulin variable domain comprises at least one CDR region and framework regions flanking the CDR, and wherein the variable domain is a variant because the CDR region has added or substituted therein at least one amino acid sequence heterologous to the CDR and/or the flanking framework regions, and wherein the presence of the heterologous sequence provides the synthebody molecule with the ability to specifically bind to the target molecule. As described herein, in exemplary embodiments, the heterologous sequence comprises the amino acid sequence of SEQ ID NO: 3 or 6.

[0026] Ex vivo (or extracoφoreal) blood treatments according to the invention can thus be used to reduce or eliminate the presence of specific target molecules. Ex vivo solid phase perfusion eliminates potential problems from anti-idiotype responses or other reactions to synthethic constructs or monoclonal antibodies that might occur by introducing such constructs or antibodies directly into the patient. Furthermore, ex vivo therapy allows instant initiation and cessation of treatment. The present invention allows the illness, disease, or condition to be managed until the tumor is localized and treated directly, e.g., by surgical removal.

[0027] Methods of ex vivo treatment include, but are not limited to, plasma perfusion, hemodialysis, and hemofiltration. The methods can be conducted on a continuous or batch basis. "Cleansed" blood or blood component may be returned to the patient concurrently with perfusion treatment or following perfusion treatment. The perfused blood or blood component may be supplemented or reconstituted with components from donated blood, artificial or synthetic components, or chemotherapeutic agents, or some combination thereof.

[0028] A typical protocol for ex vivo perfusion would comprise selecting a patient who suffers from an adenoma, in particular an adenoma patient exhibiting one or more symptoms caused by an elevated level of a peptide hormone such as VIP, PTH, insulin, or somatostatin, and conducting ex vivo perfusion on the blood or blood component collected from the patient to remove the excess peptide hormone. The perfusion is carried out using a synthebody binding member that specifically removes the peptide hormone present in the patient's blood. The ex vivo perfusion takes place over a period of 2-6 hours, and can be repeated as necessary. Typically, the ex vivo perfusion is conducted once a day or 2-5 times per week for as long as necessary for: (i) at least one relevant symptom to abate; or (ii) for the level of peptide hormone in the blood to return to a normal level; or (iii) until the tumor has been surgically removed, or some combination thereof. A symptom of excess peptide hormone in an adenoma patient or other patient may be, but is not limited to, a higher-than- normal level of calcium in plasma, bone resoφtion, bone demineralization, hypoglycemia, diabetes, diarrhea, gallbladder dysfunction, or another known symptom resulting from an excess level of a specific peptide hormone.

[0029] The efficacy of the treatment can be determined by evaluating the symptoms of the patient and/or by measuring the concentration of target molecule in the patient's blood. Blood measurements typically involve taking a blood sample, separating serum from red blood cells, and using radioimmunoassay, enzyme linked immunosorbent assay (ELISA) or chromatographic analysis of the peptide contents in serum.

[0030] The term "hemofiltration" refers to a process of filtering blood by a membrane resulting in the separation of all proteins larger than the effective pore size of the membrane from retained plasma water and solute (these return to the patient) from the ultrafiltrate.

[0031] The term "ultrafiltrate" refers to the filtered plasma water and solute and molecules smaller than the effective pore size of the membrane.

[0032] The term "hemofilter" refers to the filter used in hemofiltration. It can be configured in a number of ways, e.g., as a series of parallel plates or as a bundle of hollow fibers. The blood path is from a blood inlet port, through the fibers or between the plates, then on to a blood outlet port. Filtration of blood occurs at the membrane with ultrafiltrate forming on the side of the membrane opposite the blood. This ultrafiltrate accumulates inside the body of the filter contained and embodied by the filter jacket. This jacket has an ultrafiltrate drainage port.

[0033] The subject or patient to which the present invention may be applicable can be any vertebrate species, preferably mammalian, including but not limited to, cows, horses, sheep, pigs, fowl (e.g., chickens), goats, cats, dogs, hamsters, mice, rats, rabbits, monkeys, chimpanzees, and humans. In a preferred embodiment, the subject is a human. The invention is particularly applicable for human subjects suffering from an excessive of peptide hormone caused by an endocrine disorder (such as, e.g„ an adenoma) that causes such an excess.

[0034] A "control" is an alternative subject or sample used in an experiment for comparison puφoses. A control can be "positive" or "negative". For example, where the puφose of the experiment or the comparison in a method is to determine a correlation of an patient treatment with a particular symptom, one may use either a positive control (a patient exhibiting the symptom and not subjected to the treatment, or a sample from such a patient), and/or a negative control (a healthy subject not subjected to the treatment). [0035] The terms "cancer," "neoplasm," and "tumor," used interchangeably and in either the singular or plural form, refer to cells that have undergone a malignant transformation that makes them pathological to the host organism. Primary cancer cells (that is, cells obtained from near the site of malignant transformation) can be readily distinguished from non-cancerous cells by well-established techniques, particularly histological examination. The definition of a cancer cell, as used herein, includes not only a primary cancer cell, but also any cell derived from a cancer cell ancestor. This includes metastasized cancer cells, and in vitro cultures and cell lines derived from cancer cells.

[0036] When referring to a type of cancer that normally manifests as a solid tumor, a "clinically detectable" tumor is one that is detectable on the basis of tumor mass, e.g., by such procedures as CAT scan, magnetic resonance imaging (MRI), X-ray, ultrasound or palpation. Biochemical or immunologic findings alone may be insufficient to meet this definition, and definitive identification of a tumor type, survival statistics and therapeutic strategies for treating a tumor are all dependant to some extent on the ability of the physician to differentiate one tumor type from another. Polyclonal or monoclonal antibodies, as well as nucleic acid probes, can be used to screen biopsy specimens to determine the derivation of a particular tumor. Effective pancreatic and neuroendocrine cancer treatment depends on the early diagnosis and identification of tumor tissue. Antibodies generated from antigen obtained from purified pancreas cell populations or antibodies directed to known polypeptides present in pancreatic cells can be used to differentiate one pancreatic tumor from another.

[0037] The meaning of the terms "about" or "approximately" will be known to those skilled in the art in light of this disclosure. Preferably, the term means within 20%, more preferably within 10%, and more preferably still within 5% of a given value or range. Alternatively, especially in biological systems, the term "about" preferably means within a factor of two of a given value.

[0038] The term "isolated" means that a polynucleotide or polypeptide molecule is separated from constituents, cellular or otherwise, in which the polynucleotide or polypeptide is normally associated in nature. For example, an isolated polynucleotide is one that is separated from the 5' and/or 3' sequences with which it is normally associated in the chromosome. As will be apparent to those of skill in the art, a non-naturally occurring polynucleotide or non-naturally occurring polypeptide, does not require "isolation" to distinguish it from any naturally occurring counteφart. A polynucleotide or polypeptide molecule that differs from its naturally occurring counteφart in terms of its primary sequence, or a polypeptide molecule that differs from its naturally occurring counteφart in terms of its glycosylation pattern need not be present in isolated form to distinguish over a product of nature.

[0039] In practicing the present invention, conventional molecular biology and microbiology techniques may be used, and these are within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (herein "Sambrook et al., 1989"); DNA Cloning: A Practical Approach, Volumes I and II (D.N. Glover ed. 1985); Oligonucleotide Synthesis (M.J. Gait ed. 1984); Nucleic Acid Hybridization (B.D. Hames & S.J. Higgins eds. (1985)); Transcription And Translation (B.D. Hames & S.J. Higgins, eds. (1984)); Animal Cell Culture (R.I. Freshney, ed. (1986)); Immobilized Cells And Enzymes (IRL Press, (1986)); B. Perbal, A Practical Guide To Molecular Cloning (1984); and F.M. Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (1994); among others.

Target Molecules [0040] As used herein, the term "target molecule" includes any molecule to be specifically removed from blood or from a blood component such as plasma. An example of such a target molecule is a peptide hormone produced in excess by an adenoma or endocrine tumor such as, e.g., insulin, PTH, glucagon, somatostatin, or VIP. A peptide is a polypeptide of less than about 50 amino acid residues, usually produced by prototypes of larger pre-pro- protein. Other target molecules can include autocrine growth factors, and peptide hormones produced by overactive, but non-malignant glandular tissues. Target molecules may be molecules that are present and detectable in the blood of normal individuals, but which are present at an excess concentration in certain subjects as the result of a disease, illness, disorder or condition. For example, the target molecule may be present in an excess concentration as the result of a subject having an adenoma or endocrine tumor. Alternatively, the target molecule may not be present or detectable in the blood of a normal individual, but only present or detectable in the blood of a subject having a particular disease, illness, disorder or condition. Where the target molecule is present, or present in an excess concentration, as the result of a subject having, e.g., an adenoma or endocrine tumor, such target molecule may be produced directly by the tumor, or alternatively may be produced by other cells or tissues in the body as a consequence of the presence of the tumor, or a combination thereof. Preferably, the target molecule is not a protein (e.g., a polypeptide of greater than about 50 amino acid residues, or a polypeptide having an amino acid sequence corresponding to an entire m-RNA coding sequence, optionally lacking a signal peptide sequence).

[0041] A target molecule may comprise a particular target sequence that is preferably unique to the target molecule, or does not occur in other serum proteins or hormones in the subject. The target sequence usually comprises the whole or a part of the sequence or domain that actually interacts with the synthebody binding partner. Examples of target molecules and target sequence are exemplified below. However, any sequence from a target molecule may be useful in the preparation of a synthebody binding partner.

[0042] An insulin target molecule may comprise an amino acid sequence selected from:

AVSEHQLLHDKGKSIQDLRRRFFLHHLIAEIHT (SEQ ID NO: 4) and EHQLLHDKGKSIQDLR-RRFFLHHLIAEIHT (SEQ ID NO:5)

[0043] Another particular target molecule may comprise the following PTH sequence:

AVSEHQLLHDKGKSIQDLRRRFFLHHLIAEIHTA (SEQ ID NO: 1)

[0044] Yet another particular target molecule is a PTH receptor agonist/partial antagonist comprising the amino acid sequence: EHQLLHDKGKSIQDLRRRFFLHHLLAEIHTA (SEQ ID NO: 2)

[0045] The amino acid sequence of human somatostatin, glucagon, VIP, and other peptide hormones are publicly available, for example, via GenBank (NCBI) and other databases. Representative GenBank accession numbers for selected peptide hormones are NP 001039 (somatostatin); XP_049901 (VIP), and 720967A (glucagon). These GenBank entries, as accessed on July 9, 2003, are hereby incoφorated by reference in their entireties.

Binding Partners [0046] "Binding partners" herein are molecules that bind with target molecules, including, but are not limited to, monoclonal antibodies, variants of immunoglobulin variable domains, modified immunoglobulins, synthebodies, endogenous or variant receptors, synthetic ligands, and the like, and which bind target molecules specifically. Preferred, although non-limiting, types of binding partners are synthebodies (see below).

[0047] The ability of the binding partner molecules useful in the present invention to "interact" with a target molecule is that these molecules are recognized by and preferably or specifically bind the target molecule. In a preferred embodiment, the binding partners useful in the present invention bind specifically to insulin or PTH peptides, and preferably to the sequences exemplified above or portions thereof.

[0048] For certain applications of immunoadsoφtion in ex vivo perfusion procedures, the binding partner is an antibody or antibody variant. The antibodies useful herein can be polyclonal or monoclonal, native or engineered in any suitable manner, and are selective for the particular target molecule. Such antibodies are conveniently made using the methods and compositions disclosed in Harlow and Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, 1988, as well as immunological and hybridoma technologies known to those skilled in the art.

[0049] The term "antibody" is intended to include immunoglobulins of all isotypes and species. The antibody can be of can be any type, e.g., an IgG, IgE, IgM, IgD or IgA, preferably, the antibody is an IgG. In another specific embodiment, the construct is derived from a T lymphocyte receptor. For antibody variants such as synthebodies, the immunoglobulin molecule modified to generate the constructs is preferably a monoclonal antibody. As described above, monoclonal antibodies are conveniently made using the methods and compositions disclosed in Harlow and Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, 1988, as well as immunological and hybridoma technologies known to those skilled in the art. Additionally, the antibody may be of any subclass or isotype of each particular class of antibodies. Particular isotypes of monoclonal antibodies can be prepared either directly by selecting from the initial fusion, or prepared secondarily, from a parental hybridoma secreting a monoclonal antibody of different isotype by using the sib selection technique to isolate class switch variants (Steplewski et al., Proc. Natl. Acad. Sci USA 1985, 82:8653; Spira et al., J. Immunol. Meth. 1984, 74:307). The antibody that is modified may be a naturally occurring or previously existing antibody, or may be synthesized from known antibody consensus sequences, or any other antibody consensus or germline (i.e., unrecombined genomic sequences) sequences (e.g., those antibody consensus and germline sequences described in Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5^th Ed., N1H Publication No. 91-3242, pp. 2147-2172).

[0050] A "monoclonal antibody" is an immunoglobulin secreted by a single clone of cells. Any technique that provides for the production of antibody molecules by continuous cell lines in culture may be used. These include but are not limited to the hybridoma technique originally developed by Kδhler and Milstein (Nature 1975, 256:495 497), as well as the trioma technique, the human B cell hybridoma technique (Kozbor et al., Immunology Today 1983, 4:72; Cote et al., Proc. Natl. Acad. Sci. USA 1983, 80:2026 2030), and the EBV hybridoma technique to produce human monoclonal antibodies (Cole et al., in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77 96, 1985). In an additional embodiment of the invention, monoclonal antibodies can be produced in germ free animals (see International PCT Publication WO 89/12690).

[0051] Fragments of an immunoglobulin family protein that are specific to a target molecule can also be prepared. For example, such fragments include but are not limited to: F(ab')₂ fragments that contain the variable regions of both the heavy and the light chains, the light constant region and the CHI domain of the heavy chain, which fragments can be generated by pepsin digestion of an antibody; Fab' fragments; Fab fragments generated by reducing the disulfide bonds of an F(ab') fragment (King et al, Biochem. J., 1992, 281 :317); and Fv fragments, i.e., fragments that contain the variable region domains of both the heavy and light chains (Reichmann and Winter, J. Mol. Biol. 1988, 203:825; King et al, Biochem J. 1993, 290:723).

[0052] Single chain antibodies (SCA) may also be prepared (U.S. Patent 4,946,778; Bird, Science 1988, 242:423 426; Huston et al, Proc. Natl Acad. Sci. USA 1988, 85:5879 5883; and Ward et al, Nature 1989, 334:544 546). Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide. Additionally, the invention also provides heavy chain and light chain dimers and diabodies.

[0053] Modified chimeric or humanized antibodies may also be prepared. A chimeric antibody is a molecule in which different portions of the antibody molecule are derived from different animal species, such as those having a variable region derived from a murine monoclonal antibody and a constant region derived from a human immunoglobulin constant region. Techniques have been developed for the production of chimeric antibodies (Morrison et al, Proc. Natl. Acad. Sci. USA, 1984, 81:6851-6855; Neuberger et al, Nature, 1984, 312:604 608; Takeda et al, Nature, 1985, 314:452-454; Oi et al, BioTechniques, 1986, 4:214; International Patent Application No. PCT/GB85/00392) by splicing the genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity. In a specific embodiment, the chimeric antibody contains the variable domain of a non-human antibody and the constant domain of a human antibody. In another embodiment, the construct is derived from a humanized antibody, in which the CDRs of the antibody (except for the one or more CDRs containing the heterologous binding sequence) are derived from an antibody of a non human animal and the framework regions and constant region are from a human antibody (see, U.S. Patent No. 5,225,539; Oi et al, supra). The creation of completely human monoclonal antibodies is possible through the use of transgenic mice in which the mouse immunoglobulin gene loci have been replaced with human immunoglobulin loci to provide in vivo affinity- maturation machinery for the production of human immunoglobulins. Synthebodies

[0054] A prefeoed, although non-limiting, type of binding partner is an antibody variant termed "synthebody" (for "synthetic antibody"). A synthebody includes an antibody variable region, and preferably also regions corresponding to an antibody constant region. The synthebody can be in the form of any immunoglobulin family protein or polypeptide, such as an antibody Fv heterodimer, an antibody tetramer, a T lymphocyte receptor heterodimer, etc. In a prefeoed type of synthebody, one or more CDR-regions contain a binding site derived from a ligand to the target molecule. International PCT Publication WO 99/25378 by Burch describes synthebodies that bind one member of a binding pair and have at least one complementarity determining region (CDR) that contains the amino acid sequence of a binding site for that member of the binding pair. Other types of synthebodies for specific binding or induction of anti-idiotype responses have been described, e.g., in International PCT publication Nos. WO 01/81579 and WO 01/88159, both by Burch et al.

[0055] A synthebody preferably comprises the naturally occurring receptor, or a part or acceptable variant of such a receptor, specific for the target molecule. In the present invention, prefeoed receptor sequences are those specific for, and binding to, insulin, PTH, glucagon, VIP, or somatostatin. Various receptors and receptor subtypes that interact with each particular target molecule are well known in the art. For example, GenBank discloses the sequences for various receptors for insulin (including Accession No. NP_000199), PTH (including Accession No. NP_000307), somatostatin (AAF42810, AAF42809, etc.), VIP (NP_003373, XP_003226, etc.), and glucagon (AAA53628, AAA35897, etc.). These GenBank entries, as accessed on July 9, 2003, are hereby incoφorated by reference in their entireties.

[0056] For example, when the target molecule is a PTH peptide comprising the amino acid sequence of SEQ ID NO: 1 or 2, a prefeoed synthebody binding partner can comprise at least 10, preferably at least 15, and even more preferably at least 20, sequential amino acids of the following amino acid sequence from the PTH receptor:

MGTARIAPGLALLLCCPVLSSAYALVDADDVMTKEEQIFLLHRAQAQ CEKRLKEVLQRPASI (SEQ ID NO:3) [0057] As described by Vilardaga et al. (Mol Endocrinol 2001 ; 15 : 1186-99), this segment of the PTH receptor sequence, located in the N-terminal section of the receptor, participates in high-affinity PTH-binding. Routine testing of synthebody variants in which the at least 10 amino acid fragments are inserted into a synthebody framework can be made to determine which synthebody variants have the most suitable binding characteristics, such as the strongest affinity, for PTH in the context of the present invention. Alternatively, one or more CDR-regions, particularly the CDR3 region, of the synthebody may comprise the entire sequence of SEQ ID NO:3.

[0058] When the target molecule is insulin, comprising the amino acid sequence of SEQ ID NO: 4 or 5, a prefeoed synthebody binding partner can comprise at least 10, preferably at least 15, and even more preferably at least 20, sequential amino acids of the following amino acid sequence from the insulin receptor:

MGTGGRRGAAAAPLLVAVAALLLGAAGHLYPGEVCPGMDIRNNLTR LHELENCSVIEGHLQILLMFKTRPEDFRDLSFPKLIMITDYLLLFRVYGL ESLKDLFPNLTVIRGSRLFFNYALVIFEMVHLKELGLYNLMNITRGSVR ffiKNNELCYLATIDWSRILDSVEDNHIVLNKDDNEECGDICPGTAKGKT NCPATVINGQFVERCWTHSHCQKVCPTICKSHGCTAEGLCCHSECLGN CSQPDDPTKCVAC (SEQ ID NO:6)

[0059] As described by Christensen et al. (J Biol Chem 2002;277:18340-5), this fragment of the insulin receptor is able to fully reconstitute the insulin binding site. Routine testing of synthebody variants in which the at least 10 amino acid fragments are inserted into a synthebody framework can be made to determine which synthebody variants have the most suitable binding characteristics to insulin in the context of the present invention. Alternatively, one or more CDR-regions, particularly the CDR3 region, of the synthebody may comprise the entire sequence of SEQ ID NO:6.

[0060] The variant of the immunoglobulin variable domain typically includes (i) at least one CDR region and (ii) framework regions flanking the CDR. The term "CDR" refers to a part of the variable region of an immunoglobulin family protein that confers binding specificity, e.g., antibody specificity for antigen. In antibodies, CDRs are highly variable and accessible. In synthebodies, the CDR region has added or substituted therein at least one binding sequence. The binding sequence is heterologous (i.e., originates from another source than the original antibody) to the CDR and preferably can bind to the target molecule to be removed from the blood or blood component. The constant region, if present, can be derived from any type of immunoglobulin molecule including, for example, but not limited to, antibodies, T lymphocyte receptors, cell-surface adhesion molecules such as the co-receptors CD4, CD8, CD 19, and the invariant domains of MHC molecules.

[0061] In one embodiment, the synthebody has a heavy chain and a light chain, wherein the heavy chain comprises (1) at least one CDR region having added or substituted therein at least one amino acid sequence which is heterologous to the CDR and includes a sequence from a target molecule-binding protein (e.g., a peptide hormone receptor), and framework regions flanking the CDR, wherein the heterologous sequence is capable of binding to the target molecule to be removed from the blood or blood component; and (2) three constant domains from an immunoglobulin heavy chain, and wherein the light chain has a second variable domain linked to the heavy chain, and a constant domain from an immunoglobulin light chain. In another embodiment, the synthebody has a heavy chain and a light chain, wherein the light chain comprises (1) at least one CDR region having added or substituted therein at least one amino acid sequence which is heterologous to the CDR, and framework regions flanking the CDR, wherein the heterologous sequence is capable of binding to the target molecule to be removed from the blood or blood component; and (2) a constant domain from an immunoglobulin light chain, and the heavy chain has a second variable domain linked to the light chain and three constant domains from an immunoglobulin heavy chain.

[0062] The variable region variants and antibodies having CDR-grafted variable regions encoded by CDR grafted variable region genes can be constructed by various methods such as site directed mutagenesis as described in Jones et al, Nature 1986, 321:522; Riechmann et al, Nature 1988, 332:323; in vitro assembly of entire CDR grafted variable regions (Queen et al, Proc. Natl. Acad. Sci. USA 1989, 86:10029); and the use of PCR to synthesize CDR grafted genes (Daugherty et al, Nucleic Acids Res. 1991, 19:2471). [0063] For example, one method for producing a nucleic acid encoding a synthebody is to modify a nucleic acid sequence that encodes an immunoglobulin superfamily molecule, e.g., an antibody molecule or at least the variable region thereof, using the "PCR knitting" approach (Figure 1). In "PCR knitting", a nucleic acid sequence, such as a consensus variable region sequence, is used as a template for a series of PCR reactions that result in the selective insertion of a nucleotide sequence that encodes the desired peptide sequence into one or more CDRs of the variable domain. Oligonucleotide primers for these PCR reactions are designed to contain regions complementary to the framework sequences flanking the designated CDR at the 3 'ends and sequences that encode the peptide sequence to be inserted at the 5 'ends. Additionally, these oligonucleotides preferably contain about ten bases of complementary sequences at their 5 'ends. These oligonucleotide primers can be used with additional flanking primers to insert the desired nucleotide sequence into the selected CDR, as shown in Figure 1, resulting in the production of a nucleic acid coding for the synthebody construct.

[0064] The ex vivo perfusion methods of the invention may use a construct that is derived from a human immunoglobulin superfamily protein, a chimeric or humanized antibody, or an immunoglobulin superfamily protein from a heterologous species such as, for example, a mouse, which may or may not be humanized. For example, CDR grafted antibodies can be generated in which the CDRs of the murine monoclonal antibody can be grafted onto the framework regions of a human antibody. Following grafting, most antibodies benefit from additional amino acid changes in the framework region to maintain affinity, presumably because certain framework residues are necessary to maintain CDR conformation, and some framework residues have been demonstrated to be part of the antigen combining site. Such CDR grafted antibodies have been successfully constructed against various antigens including, for example, antibodies against IL 2 receptor, as described in Queen et al (Proc. Natl. Acad. Sci. USA 1989, 86:10029), antibodies against cell surface receptors CAMPATH, as described in Riechmann et al. (Nature, 1988, 332:323); antibodies against hepatitis B, as described in Co et al. (Proc. Natl. Acad. Sci. USA 1991, 88:2869); and antibodies against viral antigens of the respiratory syncitial virus, as described in Tempest et al. (BioTechnology 1991, 9:267). Thus, in specific embodiments of the invention, the antibody construct comprises a variable domain in which at least one of the framework regions has one or more amino acid residues that differ from the residue at that position in the naturally occurring framework region. The techniques employed in creating CDR grafted antibodies can be adapted for use in preparing synthebody binding partners useful in practicing the present invention.

[0065] In one preferred embodiment, the invention uses, as a binding partner, a synthebody or fragment thereof, that interacts with insulin, as described above. In another prefeoed embodiment, the invention uses a synthebody, or fragment thereof, that interacts with PTH as described above. In yet another preferred embodiment, the invention uses a synthebody, or fragment thereof, that interacts with somatostatin, NIP, or glucagon as described above.

[0066] The binding partner can also be modified, e.g., by the covalent attachment of any type of molecule, as long as such covalent attachment does not prevent or substantially inhibit a functional interaction between the synthebody and the target molecule. For example, but not by way of limitation, the synthebody binding partner may be modified by glycosylation, acetylation, PEGylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, or linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications may be carried out by known techniques including, but not limited to, specific chemical cleavage, acetylation, formulation, metabolic synthesis of tunicamycin, etc.

Ex Vivo Perfusion Device [0067] In the ex vivo perfusion device, the binding partner molecules are immobilized, i.e., fixed so that neither the binding partner, nor the binding pair, travel with the blood. Preferably, the binding partner is immobilized on a solid support using covalent or affinity binding. Covalent linkage can be achieved using standard cyanogen bromide (CΝBr) or other activation techniques (International PCT publication WO 00/74824 by Bristow, European Patent No. 272 792 to Jones, U.S. Patent No. 5,122,112 to Jones), or a high affinity interaction, such as that between avidin and biotin (International PCT publication WO 00/74824 by Bristow; U.S. Patent No. 6,251,394 to Nilsson). An antibody or synthebody binding partner can be attached via its Fc region, if present, to a protein- A column (Kiprov et al, J. Biol. Res. Mod. 1984, 3:341-346; Jones et al, J. Biol. Res. Mod. 1984, 3:286-292; Besa et al, Am. J. Med. 1981, 71:1035-1040; EP Application 172018 of Bensinger; EP Application 079221 of Terman; and U.S. Patent No. 4,614,513 to Bensinger).

[0068] In one embodiment, the ex vivo perfusion device is designed as a hemofiltration device with hemofiltrate outflow connecting to a solid state perfusion device, and with the perfusate rejoining red blood cell-rich efflux from the hemofiltration device. The perfusion device can be designed to have plates inserted in the direction of the flow or to have hollow fibers in the direction of flow, or to have concentric cylinders inserted in the direction of flow.

[0069] The solid support utilized in ex vivo perfusion devices and methods can be made out of any of a variety of substances (e.g., nitrocellulose, cellulose, nylon, plastic, rubber, polyacrylamde, agarose, or poly(vinylalcoholo-co-ethylene), and can be formed into a variety of shapes, including flat dialyzers, semi-permeable membranes, semi-permeable hollow fibers, coils, permeable spheres, dialysis membranes, and plasmapheresis filters, optionally using linker molecules such as PEG (polyethelene glycόl) to attach the binding partner (see International PCT publication WO 00/74824 by Bristow). In a hemofiltration device, the solid support may be, for example, in the form of beads, plates, hollow filters, or any combination thereof. An instructive method designed to remove small, non-protein- bound toxins using hollow-fiber technology is disclosed in U.S. Patent No. 5,919,369 to Ash.

[0070] The binding partners described herein can be used in amounts sufficient to remove the target molecule virtually completely from the blood or blood component, or simply to reduce the amount of the target molecule in the blood or blood component to a level that is normal or less than normal. The precise amount of the binding partner to be employed depends on the efficiency of the apparatus used, and the amount of target molecule in the blood. The amount of binding partner to be immobilized to the solid support can vary depending on the affinity between the binding partner and target molecule, the type of perfusion device, and the length of time of the perfusion treatment, among other factors. This amount can be determined empirically, according to the judgment of the practitioner and each patient's circumstances, and according to both standard clinical techniques and the results of published clinical trials. Typical parameters to consider are the level of target molecule in the blood of the patient, and the affinity of the synthebody to the target molecule. For example, the amount of immobilized binding partner can be in the range of 10¹ to 10⁵⁰, preferably 10⁸ to 10²⁰, and even more preferably 10⁸ to 10¹² target molecule binding sites per perfusion device. In one embodiment, the amount of an insulin-specific synthebody to be attached to a solid phase such as polypropylene would typically be the amount necessary to remove approximately 1 μg/ml insulin from the blood of the patient, cooesponding to a total of approximately 8 grams of insulin per adult (assuming about 8 liters of blood per patient). The same type of calculation can be made for other target molecules.

[0071] Various designs for hemodialysis and ex vivo perfusion devices are known, and have been described, e.g., in patent literature (see, e.g., U.S. Patent Nos. 4,252,653 to Beck et al; 4,824,432 to Skurkovich; 5,122,112 to Jones; 5,919,369 to Ash; and 6,287,516 to Matson; and International PCT publications WO 90/15631 by Ahmad et al; WO 00/74824 by Bristow; WO 01/37900 by Burbank et al. ; and WO 01/45769 by Burbank. Based upon the present disclosure and the level of knowledge in the art, the skilled artisan can design a perfusion device comprising synthebodies for use in the present invention.

[0072] The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and the accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

[0073] Patents, applications, publications, procedures, and literature references cited herein are incoφorated herein by reference in their entireties.

Claims

WHAT IS CLAIMED IS:

1. A method of removing a target molecule from blood or a blood component, which method comprises contacting ex vivo the blood or the blood component containing an undesirable level of the target molecule with an immobilized synthebody specific for the target molecule under conditions that result in the binding of the target molecule to the synthebody.

2. The method of claim 1 wherein the target molecule is produced in a subject as a consequence of an illness, disease or condition.

3. The method of claim 2, wherein the illness, disease or condition is a tumor

4. The method of claim 3, wherein the tumor is an adenoma.

5. The method of claim 1 wherein the target molecule is constitutively present at a level lower than the undesirable level in blood of a normal subject.

6. The method of claim 1 wherein the target molecule is not present in blood of a normal subject.

7. The method of claim 1 wherein the target molecule is a peptide.

8. The method of claim 7 wherein the peptide is a member selected from the group consisting of insulin, parathyroid hormone (PTH), glucagon, somatostatin, and vasoactive intestinal peptide (VIP).

9. The method of claim 8 wherein the peptide is PTH.

10. The method of claim 8 wherein the peptide is insulin.

11. The method of claim 3 wherein the tumor is a PTH-secreting parathyroid adenoma.

12. The method of claim 3 wherein the tumor is an insulin-secreting pancreatic insulinoma.

13. The method of claim 1 wherein the contacting comprises perfusion.

14. The method of claim 13 wherein the perfusion is carried out with an assisting pump.

15. The method of claim 1 wherein the synthebody is immobilized on a solid support.

16. The method of claim 15 wherein the solid support is incoφorated in a hemofiltration device.

17. The method of claim 15 wherein a second synthebody specific for a different target molecule is immobilized on the support.

18. The method of claim 15 wherein the solid support comprises beads.

19. The method of claim 15 wherein the solid support comprises plates.

20. The method of claim 15 wherein the solid support comprises hollow filters.

21. The method of claim 1 wherein the synthebody comprises a variant of an immunoglobulin variable domain, the immunoglobulin variable domain comprising: (i) at least one CDR region and (ii) framework regions flanking the CDR, wherein the variant comprises the CDR region having added or substituted therein at least one amino acid sequence which is heterologous to the CDR and the flanking framework regions, and the heterologous sequence is capable of specifically binding to the molecule.

22. The method of claim 21, wherein the heterologous sequence comprises at least 10 sequential amino acid residues from SEQ ID NO: 3 or 6.

23. The method of claim 21 wherein the immunoglobulin is an antibody.

24. An apparatus comprising a hemofiltration device comprising a solid support having a synthebody binding partner of a peptide hormone immobilized thereron.

25. A method of treating an adverse effect caused by a parathyroid hormone adenoma in a patient in need of such treatment, the method comprising removing a parathyroid hormone produced by the adenoma from the blood of the patient by contacting ex vivo the blood or a blood component with an immobilized synthebody binding partner of the hormone.

26. The method of claim 25 wherein the synthebody binding partner is specific for a parathyroid hormone comprising an amino acid sequence as set forth in SEQ ID NO: 1 or 2.

27. The method of claim 26 wherein the synthebody binding partner comprises at least 10 sequential amino acid residues of SEQ ID NO: 3.

28. A method of treating an adverse effect caused by a pancreatic insulinoma in a patient in need of such treatment, the method comprising removing insulin produced by the pancreatic insulinoma from the blood of the patient by contacting ex vivo the blood or a blood component with an immobilized synthebody binding partner of the insulin.

29. The method of claim 28 wherein the synthebody binding partner is specific for an insulin sequence comprising at least one amino acid sequence as set forth in SEQ ID NO: 4 or 5.

30. The method of claim 29 wherein the synthebody binding partner comprises at least 10 sequential amino acid residues of SEQ ID NO: 6.

31. A method of treating hypeφarathyroidism in a patient in need of such treatment, the method comprising removing a parathyroid hormone from the blood of the patient by contacting ex vivo the blood or a blood component with an immobilized synthebody binding partner of the hormone.

32. The method of claim 31 wherein the hormone comprises the amino acid sequence of SEQ ID NOS: 1 or 2.

33. The method of claim 32 wherein the binding partner comprises at least 10 sequential amino acid residues of SEQ ID NO: 3.

34. The method of claim 33, wherein the binding partner comprises SEQ ID

NO:3.