The present invention relates to a molecule binding to soluble tumor necrosis factor receptor (sTNFR) for use in a method for sensitizing a tumor cell in a patient to a treatment with a chemotherapeutic agent, wherein said molecule is immobilized on a solid support that is contacted with a body fluid of said patient. Furthermore, the present invention refers to a method for sensitizing a tumor cell and to a body fluid from which sTNFR has been removed for use in a method for sensitizing a tumor cell in a patient to a treatment with a chemotherapeutic agent. Moreover, the present invention refers to a chemotherapeutic agent for use in a method for treating a tumor in a patient for sensitizing a tumor cell of said tumor in said patient to a treatment with said chemotherapeutic agent.

In the developed countries, cancer is one of the major causes of death throughout the population. Therefore, various therapeutic methods have been developed since decades. However, so far, no therapeutic approach is fully satisfying. Today, cancer therapy is mainly based on chemotherapy, targeted small molecules, radiation therapy, surgery, immunotherapy, monoclonal antibody therapy and/or other methods.

Conventional chemotherapeutic cancer therapy is based on the use of comparably high concentrations of agents that have cytotoxic effects predominantly on neoplastic cells. The predominant killing of neoplastic cells occurs due to the fact that cancer cells replicate faster than most other cells of the adult human body. However, it is widely known that due to the high concentrations of chemotherapeutic agents required for effective chemotherapeutic treatments, also other cell types replicating relatively fast, such as, e.g., hair follicle cells and mucosa cells, are severely impaired. Therefore, the patient is exposed to undesired side effects. Adverse effects related to the chemotherapeutic treatment can lead to the premature discontinuation of the therapeutic treatment. In some instances chemotherapy can even cause the death of the patient.

Alternatively or additionally, surgery and radiation are used to reduce tumor bulk. Success rates for these therapeutic approaches are limited due to the inoperability of several organs or sites of the human body, insensitivity of organs to radiation, or acquired radioresistance of primary or metastatic tumor lesions. A surgical procedure may even require the amputation of a limb, a breast, a testicle, an inner organ, a part of the skin and/or parts of one or more of the aforementioned. Further, a surgical procedure may lead to an artificial anus and/or to a bladder catheter. Likewise, radiation may lead to severe symptoms of intoxication.

Other treatments have been tried in an attempt to improve mortality and morbidity. Today, some types of cancer can be treated by immunotherapy (e.g., therapeutic vaccines such as sipuleucel-T, or cytokine-based immunotherapy (e.g. Interleukin-2, Interferon-alpha)), monoclonal antibody therapy (e.g. bevacizumab), targeted small molecules (e.g., imatinib) and/or other methods, such as, e.g., cryoablation or isolated organ perfusion. These treatments may bear significant advantages in comparison to the conventional chemotherapeutic treatments described above. However, many of these treatments have limited efficacy, are associated with severe toxicities, and/or can only be used for one or few types of cancer. Therefore, there are numerous types of cancer which can, so far, only be treated with conventional cytotoxic agents, or which cannot be treated effectively at all. Irrespective of recent advances there remains a high unmet medical need associated with common malignancies with high mortality, high morbidity, high rates of recurrence, short periods of disease- or progression-free survival, rapid tumor growth, and high risk of formation of (micro-) metastases.

The addition of cytokines, alone or in combination, such as, e.g., tumor necrosis factor alpha (TNF-alpha), interferon gamma (IFN-γ), and interleukin-2 (IL-2) have been used clinically, but have led to durable remissions only in small numbers of patients treated and produced no significant clinical responses in overall target populations.

A still further therapeutic strategy is the removal of molecules based on the molecular weight by means of ultrapheresis (therapeutic apheresis, plasmapheresis) promoting an immune attack on the tumors by the patient's own white blood cells (cf, US 4,708,713). In particular, it has been demonstrated that the removal of components present in the blood having a molecular weight of 120,000 Da (dalton) or less can lead to an induction of an immune response against transformed, infected or diseased tissue (cf, US 6,620,382). However, the unspecific removal of a large fraction of components having a molecular weight of 120,000 Da may lead to significant disadvantages as also molecules that have positive effects on the patient's vitality such as, e.g., TNF- , IL-2, or IFN-γ are removed from the high-molecular weight components containing, e.g., antibodies. A patient may also be treated by the selective removal of one or more cytokine receptors, wherein it is intended to provoke and/or stimulate the patient's immune response against the tumor cells. However, today, for a majority of patients, the most promising therapy is still the treatment by means of a chemotherapeutic agent or by means of a combination of several chemotherapeutic agents. In this context, a well-known problem is that the required concentrations of chemotherapeutic agents are so high that considerable undesired side effects inevitably occur the patient severely suffers from. Moreover, during the treatment, many patients become increasingly insensible to the agent and would require increasing amounts thereof to achieve the therapeutic aim up to concentrations that are not tolerable any more.

Therefore, there is still a need for improved cancer therapeutic means that allow usage of lower concentrations of chemotherapeutic agents and nevertheless show comparable efficiency of the therapy.

Surprisingly, it could be demonstrated that the removal of sTNFR2 alone or together with one or more cytokine receptors sensitizes tumor cells to a treatment with a chemotherapeutic agent allowing the decrease of the required amount of said chemotherapeutic agent.

In a first aspect, the present invention refers to a molecule binding to soluble tumor necrosis factor receptor 2 (sTNFR2) for use in a method for sensitizing a tumor cell in a patient to a treatment with a chemotherapeutic agent, wherein said molecule is used to remove sTNFR2 from a body fluid of said patient.

Preferably, said molecule is immobilized on a solid support that is contacted with a body fluid of said patient.

When the molecule binding to sTNFR2 is contacted with a body fluid of said patient, this will lead to a removal of sTNFR2 from said body fluid and thereby decrease the concentration of sTNFR2 in the body fluid. Most preferably, the concentration of sTNFR2 is decreased in blood.

As used herein, the term "soluble tumor necrosis factor receptor 2 (sTNFR2)" refers to a polypeptide that has a sequence identity of at least at least 75%, even more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, and most preferably 100% of the sequence of SEQ ID NO: 1. In the art, several designations are common for sTNFR2, such as, e.g., "soluble tumor necrosis factor receptor 2", "soluble tumor necrosis factor receptor type II", "soluble tumor necrosis factor receptor IB", "soluble tumor necrosis factor receptor superfamily member I B", "soluble tumor necrosis factor receptor superfamily IB", "soluble tumor necrosis factor-binding protein 2", "sTNF-alpha receptor", "TNFR1B", "sTNFR-II", "TNFRSFIB", "sTBP-2", "sTBP-II", "sTBP2", "sTBPII", "p75", "p80", "sTNF-R2" or "CD 120b". It will be understood that these names may be understood interchangeably.The sTNFR2 polypeptide may bear the property to bind to tumor necrosis factor (TNF). TNF may be, e.g., TNF-alpha. As used herein, the terms "TNF", "cachexin" or "cachectin", TNF-a and "tumor necrosis factor-alpha" may be understood interchangeably. In nature, sTNFR2 may be generated by shedding, thus, by proteolytic cleavage of the full-length TNFR2 associated with the membrane. Typically, shedding will be executed by one or more matrix metalloprotease(s).

In the blood stream, one of the major functions of sTNFR2 is capturing TNF and, thereby, lowering the TNF concentration locally in the proximity of the cells producing sTNFR2 and systemically in the entire blood stream. A molecule structurally and functionally related to sTNFR2 is sTNFRl . In fact, also sTNFRl is released from the cells and is able to lower the TNF concentration locally in the proximity of the cells producing sTNFRl and systemically in the entire blood stream. Therefore, the person skilled in the art will notice, that instead of sTNFR2, also sTNFRl may be used in the context of the present invention.

Accordingly, the present invention also refers to a molecule binding to soluble tumor necrosis factor receptor 1 (sTNFRl) for use in a method for sensitizing a tumor cell in a patient to a treatment with a chemotherapeutic agent, wherein said molecule is used to remove sTNFRl from a body fluid of said patient.

As used herein, the term "removal" may be understood in the broadest sense as the decrease of the concentration of a soluble cytokine receptor in the body fluid. As used herein, the term "decrease", "lower" and "deplete" may be understood interchangeably. After removal, the concentration of the soluble cytokine receptor may be decreased to less than 50%, less than 25%, less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, or less than 0.1 % of the initial concentration. The initial concentration may be the concentration found in any fluid, in particular the concentration naturally found in a body fluid.

As laid out in detail below, the molecule binding to sTNFR2 or to at least one other soluble cytokine receptor (see below) may be any molecule binding to the soluble cytokine receptor of interest with a dissociation constant (Kd) of less than 10 mM, less than 1 mM, less than 100 μΜ, less than 10 μΜ, less than 1 μΜ, less than 100 nM, or less than 10 nM. As used in the context of the present invention, the molecule is immobilized on a solid support.

As used herein, the term "immobilized to a solid support" means that the molecule is bound to any solid material. Herein, the term "bound" means that the molecule is covalently or quasi-covalently bound. A quasi-covalent bond is a non-covalent bond with a binding affinity with a Kd of less than 50 nM, such as, e.g., the dihydrofolate-methotrexate interaction, or the streptavidin-biotin interaction. The molecule binding to the soluble cytokine receptor may be immobilized directly or via linker. For instance a bispecific linker can bind to the surface of the solid support with one active group whereas it binds to the molecule binding to the soluble cytokine receptor with another active group. For instance, a glass surface of a solid support may be activated by silane conditioning as known in the art. Then, the surface of the solid support may further be conjugated with bispecific linker carrying, e.g., a maleimide or a succinimidyl ester (e.g., N- hydroxysuccinimidyl (NHS) esters) on the other end. A succinimidyl ester can bind to free amino groups of the molecule binding to the soluble cytokine receptor, a maleimide preferably binds to free thiol groups of the molecule binding to the soluble cytokine receptor. An active ester such as an acid halogenide (e.g., an acid chloride or an acid bromide) may bind to free amino groups or free hydroxy 1 groups of the molecule binding to the soluble cytokine receptor. Thereby, the molecule binding to the soluble cytokine receptor is immobilized on the surface of the solid support. Alternatively, antibodies binding to the molecule binding to the soluble cytokine receptor can be immobilized on the surface of a solid support. Then, in a second step, a fluid containing the molecule binding to the soluble cytokine receptor is incubated with the antibody-coated surface. As used herein, the term "coated" may be understood in the broadest sense as the conjugation of any molecular structure(s) to the surface of the solid support.

In this context, the term "tumor cell" may be understood in the broadest sense as any cell from neoplastic tissue. The neoplastic tissue may be benign or malignant and may be of any tissue origin. Preferably, the neoplastic tissue is malignant, semi-malignant or is at risk of being malignant (pre-malignant). The neoplasia may be a palpable and tactile lump, may or may not be detectible by molecular imaging, or may be a microscopic lesion of a single cell or few cells such as, e.g., a metastasis. The tumor cell may be a cell of a primary tumor or a secondary tumor. As used herein, the terms "tumor", "cancerous swelling", "neoplasia", "lesion" and "carcinoma in situ" ^' ' may be understood interchangeably. The tumor cell may origin from any tissue that can develop a tumor, such as, e.g., sarcoma (e.g., myxosarcoma, histiocytoma, liposarcoma, chondrosarcoma, osteosarcoma, angiosarcoma, leiomyosarcoma, rhabdomyosarcoma, fibrosarcoma, synovialcarcinoma, Ewing sarcoma, hemangiosarcoma, lymphangiosarcoma, adenosarcoma, carciosarcoma), bronchiocarcinoma, prostate carcinoma, cervix carcinoma, ovarian carcinoma, mamma carcinoma, bronchial carcinoma, melanoma, esophagus carcinoma, rectal carcinoma, pancreas carcinoma, bladder carcinoma, kidney carcinoma, blastoma (e.g., hepatoblastoma, retinoblastoma, nephroblastoma, neuroblastoma, medulloblastoma, glioblastoma), head and neck tumor, cerebral tumor, lymphoma (e.g., Hodgkin lymphoma, Non-Hodgkin lymphoma, multiple lymphoma, malignant lymphoma), fibroma (e.g., adenofibroma, neurofibroma), leukemia (e.g., acute myeloic leukemia, chronic myeloic leukemia), endothelioma (e.g., hemangioendothelioma), adenoma (e.g., fibroadenoma), semioma, dysgerminoma, luteoma, mesenchymal tumors, granulose cell tumor, ymphangioma, meningeoma, mesothelioma, schwannoma, chorionepitheliom, hepatoma, leukemia, teratoma, basaliom, neurodoctrine tumors, insulinoma, pheochromocytoma, melanoma, astrocytoma, anaplastic meningeoma, carcinoides, and/or (aggressive) fibromatosa.

The tumor cell may be located in any organ, such as, e.g., brain, eye, skin, lung, liver, oral cavity, pharynx, stomach, small intestine, duodenum, colon, urinary bladder, kidney, gall bladder, pancreas, vaginal tract, testicles, breast, a muscle, blood, lymph, a lymph node, esophagus, heart, a bone, bone marrow, spleen, a blood vessel, a lymphatic vessel, cervix, uterus, spinal cord, or an ovary or may be located in connective tissue, mucosa tissue, or nerve tissue. The tumor cell may also be a single cell or an aggregate of or few cells floating through the patient's body such as, e.g., via the blood stream or the lymphatic system. The term "blood stream" as used herein may be understood in the broadest sense as circulating blood. The blood stream may be in a vessel of a patient or may be in a tube, a filter, a column or any other device outside of the body.

As used herein, the term "sensitizing a tumor cell" means that the tumor cell is rendered more sensitive to a treatment with a chemotherapeutic agent. According to the present invention, this may be mainly achieved by removing at least one soluble cytokine receptor from a body fluid of the patient. The therapeutic potency of the chemotherapeutic agent with respect to the tumor cells subjected to the sensitization according to the present invention is increased compared to congeneric tumor cells without said sensitization. That means that the concentration of the chemotherapeutic agent may be decreased whereas the therapeutic effect with respect to the tumor cells of interest is extensively maintained or even increased. Depending on the chemotherapeutic agent and the type of the removed soluble cytokine receptor and the degree of removal, the required concentration to maintain the therapeutic activity may be decrease by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or even 90% or more of the concentration required without the removal of the soluble cytokine receptor.

As laid out in detail below, the chemotherapeutic agent as used herein may be any chemotherapeutic agent known in the art, preferably an alkylating agent, more preferably a nitrogen mustard alkylating agent, in particular Melphalan.

The term "body fluid" may be understood in the broadest sense as any fluid obtainable from a patient's body any fluid obtainable from a body after one or more processing step(s). Preferably, the body fluid is blood, blood plasma, blood serum, a blood fraction or lymph. More preferably, the body fluid is blood obtained from a patient.

As used herein, the term "blood" may be understood in the broadest sense as blood of a human or an animal. Preferably, the blood is the blood of the patient who receives the blood, plasma or blood fraction of which the soluble cytokine receptor has been removed. Most preferably, the patient will retrieve his own blood (autologous blood) that is returned into his blood stream after being treated by means of the molecule and/or the method of the present invention. Typically, the blood is withdrawn from the patient, treated by means of the molecule and/or the method of the present invention and, subsequently, immediately conducted back into the patient's blood stream. However, the patient's autologous blood may also be stored before treatment and/or after treatment and conducting it back into the patient's blood stream. Alternatively, the blood may also be blood originating from another individual (heterologous blood) or a mixture of autologous and heterologous blood.

As used herein, the term "plasma" may be understood in the broadest sense as blood from which the cells (e.g., red blood cells (RBCs) and leucocytes) as well as the platelets have been removed. Preferably, the plasma is obtained from the blood of the patient who receives the plasma, blood or blood fraction of which the soluble cytokine receptor has been removed.

As used herein, the term "blood fraction" may be understood in the broadest sense as any fraction of blood from which one or more factors have been removed. Exemplarily, the RBCs, some types of or all leucocytes, platelets, and/or one or more proteins may be removed. Preferably, the term "blood fraction" refers to (blood) serum, thus, plasma of which further one or more coagulation factor(s) has/have been removed. Preferably, in serum, at least fibrin and/or fibrinogen has been removed. A blood fraction may also be plasma or serum of which further proteins of a certain molecular weight range have been removed. This can be proteins of a molecular weight above a certain threshold or below a certain threshold, preferably proteins above a certain threshold are removed, more preferably proteins of more than 50,000 Da, more than 100,000 Da, more than 120,000 Da, more than 150,000 Da or more than 200,000 Da. Even more preferably, proteins of a molecular weight of more than 120,000 Da may be removed from the blood fraction. Alternatively, sTNFR2 may also be removed from any other body fluid such as, e.g., lymph (endolymph and/or perilymph), cerebrospinal fluid {liquor cerebrospinalis).

As used in the context of the present invention, the term "patient" may be understood in the broadest sense as any subject or individual bearing a tumor cell treated or to be treatment with a chemotherapeutic agent. The patient may be any animal, preferably a mammal, in particular a human. Preferably, the patient is a human suffering from a malignant neoplasia, including cancer, or a benignant neoplasia. As used herein, the term "suffering from" may be understood in the broadest sense as having a certain disease. The patient may or may not bear any symptoms of said disease. The disease may be any disease, in particular a tumorous disease, cancer. The patient may or may not show any further symptoms caused by increased soluble cytokine receptor level such as faster tumor growth, depleted immune response against tumorous or neoplastic tissue.

Highly preferably, the patient is a cancer patient. The patient may have or may be free of clinical symptoms occurring from said neoplasia. The patient may also be a patient who had a malignant or benignant neoplasia that has already been removed by any means (e.g., by surgical means, radiation and/or chemotherapeutic means) who is subjected to an after- treatment with a chemotherapeutic agent. Preferably, the patient has increased blood levels of one or more cytokine receptor(s). Most preferably, the patient is a cancer patient having increased blood levels of one or more cytokine receptor(s).

As indicated above, a molecule binding to at least one soluble cytokine receptor may be any molecule binding to the soluble cytokine receptor of interest with a dissociation constant (Kd) of less than 10 mM, less than 1 mM, less than 100 μΜ, less than 10 μΜ, less than 1 μΜ, less than 100 nM, or less than 10 nM. The molecule may be a small molecule (low-molecular weight compound) having a molecular weight of not more than 5000 Da or may be a high- molecular weight compound having a molecular weight of more than 5000 Da. Preferably, the molecule is a high-molecular weight compound. Even more preferably, the molecule is a polypeptide. As used in the context of the present invention, the term "polypeptide" may be understood in the broadest sense as a linear or branched polymer mainly composed of amino acids. A polypeptide may be a protein. The polypeptide may further contain posttranslational modifications. Posttranslational modifications are well-known in the art and may comprise but may not be limited to lipidation, phosphorylation, sulfatation, glucosylation, truncation, cyclization of several amino acid moieties, cyclization of the polypeptide strand, oxidation, reduction, decarboxylation, acetylation, amidation, deamidation, nitration, nitrosylation, disulfide bond formation, pyroglutamate formation, amino acid addition, cofactor addition (e.g., biotinylation, heme addition) and complexation of metal ions, non-metal ions, peptides or small molecules and addition of iron-sulfide clusters. Further, co-factors, such as, e.g., ATP, ADP, NAD+, NADH+H ⁺ , NADP ⁺ , NADPH+H ⁺ , metal ions, cations, anions, lipids may be bound to the polypeptide, irrespective on their biological impact. Moreover, the protein may be elongated by one or more amino acids.

Even more preferably, the molecule is selected from the group consisting of: an antibody, a cofactor, a cytokine, a synthetic polypeptide, human LDL receptor, lipocalin or fibronectin and/or a small molecule. Even more preferably, the molecule is an antibody. The antibody may be a polyclonal or a monoclonal antibody. The antibody may also be a single domain antibody (sdAB) such as, e.g., a camelid antibody (e.g., a llama antibody). As used herein, the terms "cameloid species antibody" and "camelid antibody" may be understood interchangeably.

As used herein, the terms "single-domain antibody" and "nanobody" may be understood interchangeably as an antibody fragment consisting of a single monomeric variable antibody domain that is able to bind to a specific antigen selectively. Typically, sdABs have a molecular weight of approximately 12-15 kDa. Exemplarily, sdAB may be obtained from camelids (e.g., dromedaries, camels, llamas, alpacas) and Chondrichthyes (cartilaginous fishes, such as, e.g., sharks, rays and skates). An alternative approach to obtain sdAB is to split the dimeric variable domains from common immunoglobulins (e.g., IgG) from a mammal into monomers.

The at least one soluble cytokine receptor may be any soluble cytokine receptor known in the art.

In a preferred embodiment, further another soluble cytokine receptor of an immunomodulatory cytokine is removed from the body fluid of said patient, preferably said receptor is a receptor of a proinflammatory cytokine, more preferably a receptor of a Thl-type cytokine.

This may be achieved by the use of a molecule binding to said soluble cytokine receptor. All embodiments described above relating to the molecule binding to sTNFR2 also apply to said molecule binding to another soluble cytokine receptor. The term "immunomodulatory cytokine" may be understood in the broadest sense as any cytokine that has an influence on the activity of the patient's immune system. The term "proinflammatory cytokine" may be understood in the broadest sense as any cytokine that has a positive influence on the patient's immune system. Exemplarily, a receptor of a proinflammatory cytokine may be a receptor of a chemokine (e.g., CCL (e.g., CCL1, CCL2/MCP- 1 , CCL3/MIP-l , CCL4/MIP-lp, CCL5/RANTES, CCL6, CCL7, CCL8, CCL9, CCL11, CCL 12, CCL13, CCL14, CCL 15, CCL 16, CCL 17, CCL18, CCL 19, CCL20, CCL21 , CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28), CXCL (e.g., CXCL1 KC, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8/IL8, CXCL9, CXCLIO, CXCL11, CXCL 12, CXCL 13, CXCL14, CXCL 15, CXCL 16, CXCL 17), CX3CL (e.g., CX3CL1), XCL (e.g., XCL1, XCL2)) , a tumor necrosis factor (TNF) (e.g., TNFA, lymphotoxin (TNFB/LTA, TNFC/LTB), TNFSF2, TNFSF4, TNFSF5/CD40LG, TNFSF6, TNFSF7, TNFSF8, TNFSF9, TNFSF10, TNFSF11, TNFSF13B, EDA), an interleukin (IL) (e.g., type I IL (e.g., γ-chain IL (e.g., IL2/IL15, IL4/IL13, IL7, IL9, IL21), β-chain IL (e.g., IL3, IL5, GM-CSF) IL6 like/gpl30 (e.g., IL6, IL1 1, IL27, IL30, IL31 (+non IL OSM, LIF, CNTF, CTF 1)), IL-12 family/IL12RBl (IL12, IL23, IL27, IL35), others (e.g., IL14, IL16, IL32, IL34)), type II IL (e.g., IL-10 family (e.g., IL10/IL22, IL19, IL20, IL24, IL26), interferon type III, IL28/IFNL2+3, IL29/IFNL1))), an interferon (IFN) (e.g., type I IFN (e.g., IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNA8, IFNA10, IFNA13, IFNA14, IFNA16, IFNA17, IFNA21, IFNB1, IFNK, IFNWl), type II IFN (e.g., IFNG))), a member of the IG superfamily (e.g., ILIA/ILIFI, IL1B/IL1F2, lRa/ILlF3, IL1F5, IL1F6, IL1F7, IL1F8, IL1F9, ILIFIO, 33/IL1F1 1, 18/IL1G), an IL-17 family member (e.g., IL17/IL25 (IL17A)), a growth factor or a hematopoietic (KITLG, colony- stimulating factor) (e.g., SPP1).

In a more preferred embodiment, the soluble cytokine receptor is selected from the group consisting of soluble tumor necrosis factor receptor 1 (sTNFRl), soluble interleukin-2 receptor (sIL2R), soluble interleukin-1 receptor (sILlR),soluble interleukin-6 receptor (sIL6R), soluble interleukin- 12 receptor (sIL12R), soluble interleukin- 18 receptor (sIL18R), soluble interleukin-23 receptor (sIL23R), soluble granulocyte macrophage colony-stimulating factor receptor (sGM-CSFR), soluble transforming growth factor beta receptor (sTGFpR) or soluble interferon gamma receptor (sIFNyR).

In an even more preferred embodiment, the soluble cytokine receptor is a soluble tumor necrosis factor receptor or a soluble interleukin receptor, more preferably wherein said soluble cytokine receptor is selected from the group consisting of:

i) sTNFRl ; and ii) sIL2R,

in case that sTNFR2 is removed.

As used herein, the term "soluble tumor necrosis factor receptor 1 (sTNFRl)" refers to a polypeptide that has a sequence identity of at least at least 75%, even more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, and most preferably 100% of the sequence of SEQ ID NO:2. In the art, several designations are common for sTNFRl , such as, e.g., "soluble tumor necrosis factor receptor 1 ", "soluble tumor necrosis factor receptor type I", "soluble tumor necrosis factor receptor 1A", "soluble tumor necrosis factor receptor superfamily member 1A", "soluble tumor necrosis factor receptor superfamily 1A", " soluble tumor necrosis factor-binding protein 1" , "soluble TNF-alpha receptor", "TNFRIA", "sTNFR-I", "TNFRSFIA", "sTBP-1", "sTBP-I", "sTBPl", "sTBPI", "p55", "p60", "sTNF-Rl", "sTNF-R55", or "CD 120a". It will be understood that these names may be understood interchangeably. In nature, sTNFRl may be generated by shedding, thus, by proteolytic cleavage of the full length TNFR1 associated with the membrane. Typically, shedding will be executed by one or more matrix metalloprotease(s).

As used herein, the terms "soluble interleukin-2 receptor alpha (sILR2)", "soluble IL-2 receptor alpha", "sIL-2R", "sIL2aR", "sIL2aR", "sIL2-R", "sIL2-RA" and "CD25 alpha" may be understood interchangeably.

Most preferably, the soluble cytokine receptor binding to the molecule of the present invention is sTNFR2. It will, however, be understood that alternatively or additionally other soluble cytokine receptors may be also used in the context of the present invention. Such methods are e.g. described in WO 05/107802.

As indicated above, the molecule binding to the soluble cytokine receptor may be any molecule binding to the soluble cytokine receptor of interest with a dissociation constant (Kd) of less than 10 mM, less than 1 mM, less than 100 μΜ, less than 10 μΜ, less than 1 μΜ, less than 100 nM, or less than 10 nM. As already laid out above, the molecule is preferably a polypeptide. However, also a small molecule may be used as the molecule of the present invention.

Preferably, the molecule binding the soluble cytokine receptor may bind to the soluble cytokine receptor with a Kd of less than 1 nM, more preferably less than 100 μΜ, even more preferably less than 10 μΜ, even more preferably less than 1 μΜ, in particular less than 100 nM. The molecule binding to the soluble cytokine receptor may be understood as any binding partner of the soluble cytokine receptor or any soluble cytokine receptor - binding molecule. Preferably, said molecule binding to the soluble cytokine receptor may have a higher binding affinity to the soluble cytokine receptor than to other proteins present in blood or plasma such as, e.g., albumins (e.g., serum albumin), globulins (e.g., immunoglobulins), other cytokines, interleukins, chemokines, fibrinogens, antitrypsins and/or complement factors. Further, said molecule binding to the soluble cytokine receptor may bear a significantly higher affinity to the soluble cytokine receptor than to other soluble cytokine receptors. Therefore, said molecule binding to the soluble cytokine receptor may bear more than 5fold, preferably more than l Ofold, more preferably more than 50fold, even more preferably more than l OOfold higher binding affinity to the soluble cytokine receptor than to any other soluble cytokine receptor. The molecules binding to the soluble cytokine receptor may be of natural or of synthetical origin. Further, the molecules binding to the soluble cytokine receptor may be conjugated or bound to other molecules that may refer to but may not be limited to one or more protein tag(s) (e.g., dihydrofo late, polyhistidine tag(s) ((poly)His tag(s)), streptavidin), one or more binding moiety/moieties comprising, e.g., biotin, methotrexate, glycocorticoid(s), one or more active ester(s) (e.g., N-hydroxysuccinimidyl (NHS) ester(s)), one or more isothiocyanate(s), one or more maleimide(s), one or more glutaraldehyde derivative(s), one or more carbodiimide derivative(s) or combinations thereof, one or more insoluble and/or soluble polymer(s) (e.g., polyethylene glycol (PEG), hydroxypropyl methacrylate (HPMA), polyethylene imine (PEI)), one or more antibody/antibodies or derivative(s) thereof, micro- or nanobeads (e.g., functionalized silica beads, polysaccharide-based beads), polymersomes, liposomes, one or more linker or spacer molecule(s) such as, e.g., peptide linker(a), a polyethylene glycol (PEG) linker(s), saccharide linker(s), fatty acid linker(s), alkyl Iinker(s), antibody linker(s), or a combination of two or more thereof. The molecule binding to sTNFR2 may be conjugated to one or more binding moieties and/or one or more linker molecules during synthesis (i.e., as fusion protein) or ex post synthesis or expression.

In a preferred embodiment, the molecule is selected from the group consisting of:

(i) an antibody, preferably a monoclonal antibody, a single domain antibody, an antibody fragment or an antibody mimetic;

(ii) a cofactor,

(iii) a cytokine, in particular tumor necrosis factor alpha (TNFSF2) or TNFSF2 mutein;

(iv) a synthetic polypeptide, in particular Staphylococcus aureus Protein A, human LDL receptor, lipocalin or fibronectin; and/or

(v) a small molecule, in particular an ensemblin, a macrocyclic scaffold, an Aptamers and/or a peptide staple; more preferably wherein said molecule is an antibody, in particular wherein said molecule is a monoclonal antibody or a single domain antibody.

However, as described above, an antibody according to the present invention may also be a polyclonal antibody, such as a polyclonal antibody binding to the soluble and the membrane associated forms of the TNFR2 concomitantly.

An antibody may be a monoclonal or a polyclonal antibody of any species or origin. It may bind to any epitope(s) comprised in the soluble cytokine receptor polypeptide of interest (e.g., linear epitope(s), structural epitope(s), primary epitope(s), secondary epitope(s)), including its posttranslational modifications. The epitope may be accessible by the antibody in the natural configuration of the soluble cytokine receptor of interest or may be a hidden epitope. Preferably, the epitope is accessible in the natural configuration of the soluble cytokine receptor of interest. The antibodies may be of natural origin, of gene technologic origin and/or of synthetical origin. Optionally, the antibody may also be a CovX antibody. Optionally, the antibody may also be a camelid antibody.

As used herein, the term "antibody fragments" may be understood in the broadest sense as any fragment of an antibody that still bears binding affinity to its target. Exemplarily, the antibody fragment may be a fragment antigen binding (Fab fragment), a truncated antibody comprising one or both complementarity determining region(s) (CDR(s)) or the variable fragment (Fv) of an antibody. The antibody fragments may be of natural origin, of gene technologic origin and/or of synthetical origin.

As used herein, the term "antibody mimetic" may be understood in the broadest sense as organic compounds that, like antibodies, can specifically bind antigens and that typically have a molecular mass in a range of from approximately 3 kDa to approximately 25 kDa. Antibody mimetics may be, e.g., Affibody molecules (Affibodies), Affilins, Affitins, Anticalins, Avimers, DARPins, Fynomers, Kunitz domain peptides, single-domain antibodies (e.g., VHH antibodies or VNAR antibodies) Monobodies, Diabodies, Triabodies, flexibodies and tandabs. The antibody mimetics may be of natural origin, of gene technologic origin and/or of synthetical origin.

The term "cofactor" may be understood in the broadest sense as a molecule binding to the soluble cytokine receptor in the natural environment to facilitate or enhance binding affinity or any other function thereof. As used herein, the term "cytokine" may be understood in the broadest as any small protein, peptide, or glycoprotein secreted in the body to signal in intercellular communication, that may bind to the soluble cytokine receptor, such as, e.g., tumor necrosis factor alpha (sTNFSF2).

As used throughout the invention, the term "mutein" refers to any mutated protein. The amino acid sequence of the mutein may comprise alterations or substitutions in the primary amino acid residues, may be truncated and/or may be elongated compared to the corresponding wildtype protein. The mutein may have more than 50%, preferably more than 60%, more preferably more than 70%, even more preferably more than 80%, even more preferably more than 90%, most preferably more than 95% sequence homology to the corresponding wildtype protein.

As used herein, the term "synthetic polypeptide" refers to any polypeptide that binds to the soluble cytokine receptor. A synthetic polypeptide may be obtained from chemical synthesis or from gene technological processes. A synthetic polypeptide may be, e.g., Staphylococcus aureus Protein A, low-density lipoprotein (LDL) receptor, lipocalin or fibronectin.

As used herein, the term "small molecule" refers to any molecule that binds to the soluble cytokine receptor that has a molecular mass of less than 5000 Da, preferably less than 2000 Da, more preferably less than 1500 Da, even more preferably less than 1000 Da, even more preferably less than 800 Da. The small molecule may be, e.g., an ensemblin, a macrocyclic scaffold (e.g., an ensemblin), an Aptamer and/or a peptide staple. Further, the small molecule may also be any other small molecule. It may be obtained from a natural source and be identified in a screening process. It will be understood by a person skilled in the art, that the small molecule may also be conjugated to a polymeric scaffold, such as, e.g., hydroxypropyl methacrylamide (HPMA), polyethylene imine (PEI), carboxymethyl cellulose (CMC), polyethylene glycol (PEG), collagen fiber, a silk fiber, an agarose fiber, or a micro- or nanobead.

Highly preferably, the molecule binding to at least one cytokine receptor is an antibody, in particular a monoclonal antibody. Most preferably, the molecule is a monoclonal antibody binding to at least one Thl -type cytokine, preferably sTNFRl , sTNFR2 and/or sIL2Rin, in particular binding to sTNFR2.

Optionally the solid support further has one or more molecules binding to other plasma protein(s) immobilized on a solid support, preferably wherein said molecules are molecules binding to cytokines, chemokines, soluble cytokine receptors, soluble chemokine receptors, other soluble decoy receptors, angiogenic factors, growth factors, and bone morphogenic factors more preferably molecules binding to soluble interleukin receptors (sILRs), in particular molecules binding to soluble interleukin-2 receptor alpha (sIL2R).

As used herein, the term "other plasma protein" may be understood in the broadest sense as any protein found in blood plasma that may modulate an immune response and/or may control cell growth, angiogenesis and/or apoptosis, except for sTNFRl . Exemplarily, said plasma protein may be a cytokine. Exemplarily, a cytokine may be an interleukin (IL) (e.g., IL-la, IL- Ι β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9 IL-10, IL-1 1 , IL-12, IL- 13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, 1L-21 , IL-22, IL-23, IL-24, IL-25, IL- 26, macrophage- inhibitory factor- 1), a lymphokine (e.g., granulocyte-macrophage colony- stimulating factor (GM-CSF), an interferon (e.g., IFN-a, IFN-β, IFN-γ), a chemokine (e.g., a CCL, a CXCL, an XCL, growth-related oncogene-alpha, CX3CL or a monocyte chemoattractant protein (MCP)). An "other plasma protein" may also be a soluble cytokine receptor (e.g., sIL2R, or the soluble interferon-gamma receptor), a colony stimulating factor (e.g., M-CSF, G-CSF), an angiogenic factor, a growth factor (e.g., TGF-beta 1 , TGF-beta 3, HGF, VEGF, basic FGF, IGF-1, IGF-2, PDGFR, EGF, CTGF), a bone morphogenic factor (e.g., osteoprotegerin, osteopontin, macrophage inhibitory cytokine- 1 other soluble decoy receptors (e.g., soluble VCAM-1 , soluble E-selectin, soluble P- selectin, soluble E-cadherin, soluble ILT-3, soluble MICA, soluble MICB, sULBP2, sICAM-1 , soluble Fas) or one or more mutein(s) of the aforementioned. In particular, the plasma protein may be a molecule binding to sIL2R.

As indicated above, the molecule binding to at least one cytokine receptor is immobilized on a solid support. This solid support may be any solid material known in the art, the molecule can be immobilized on in a way the molecule extensively maintains its binding activity to the soluble cytokine receptor.

In a preferred embodiment, the solid support forms part of an extracorporeal device through which the patient's body fluid can be circulated prior to being returned into said patient or forms part of an implant, in particular wherein said solid support is an extracorporeal device comprising at least one of the following:

(i) a column, in particular an adsorbent column;

(ii) a filter, in particular a capillary membrane filter with a pore size of between about 0.04 and 0.05 μιη or a parallel plate filter with a pore size of between about 0.04 and 0.08 μπι; (iii) semi-permeable material, preferably a membrane, in particular a dialysis membrane;

(iv) a bead, in particular a microbead or a nanobead; and/or

(v) the surface of said device, in particular the surface of a tubing of said device.

As used herein, the term "extracorporeal device" may be understood in the broadest sense as any device that is used ex vivo, thus, outside of the living body of the patient. The extracorporeal device may be a column, optionally coated with a molecule binding to the soluble cytokine receptor and/or filled with a column matrix coated with a molecule binding to the soluble cytokine receptor, a filter system coated with a molecule binding to the soluble cytokine receptor, a membrane, in particular a dialysis membrane, coated with a molecule binding to the soluble cytokine receptor, a tubing coated with a molecule binding to the soluble cytokine receptor, or any other device coated having a molecule binding to the soluble cytokine receptor immobilized on a solid support.

The extracorporeal device may get in contact with the body fluid containing the soluble cytokine receptor directly. Alternatively, the body fluid containing the soluble cytokine receptor may first be subjected to a previous purification step and may subsequently get in contact with the body fluid containing the soluble cytokine receptor. For instance, the suspended or emulsified cells of a body fluid such as, e.g., blood cells, may first be removed from the body fluid. Then, blood plasma is generated. The blood plasma may get in contact with the extracorporeal device that enables the removal of the soluble cytokine receptor. Subsequently, the cells may optionally be remixed with the plasma or returned into the patient separately. The separation of cells and body fluid may be used to protect the column(s), the filter or membrane device from being clogged. Likewise, a size exclusion filter may be suited upstream the extracorporeal device that enables the removal of the soluble cytokine receptor. This filter may be used to protect a column, filter or membrane device from being clogged.

As used herein, the term "get in contact" may be understood in the broadest sense as exposing the molecules binding to the soluble cytokine receptor to the body fluid, enabling the soluble cytokine receptor to bind to the molecules binding to the soluble cytokine receptor.

Alternatively or additionally, one or more type(s) of molecule(s) or one or more type(s) of cell(s) may be removed by other means such as, e.g., affinity chromatography, anion exchange chromatography, cation exchange chromatography, hydrophobicity chromatography, or electrophoresis. This process may be conducted upstream and/or downstream to the removal of the soluble cytokine receptor or may be conducted on the same solid support as the removal of the soluble cytokine receptor.

The extracorporeal device may be used offline, online or a combination thereof. As used herein, the term "offline" may be understood in the broadest sense as a system that is used batch-wise. Therefore, a certain volume of the body fluid extracted from a patient, is exposed to the device of the present invention and the soluble cytokine receptor is removed. Subsequently, the body fluid, from which the soluble cytokine receptor has been removed, is further analyzed and/or is optionally returned to the patient from whom the fluid has been obtained or to one or more other patient(s), in particular other patient(s) of the same patient as the graft patient the body fluid is obtained from. As used herein, the term "online" may be understood in the broadest sense as a system that is used in a continuous flow and/or is connected with the patient's blood or lymphatic vessel(s). Highly preferably, the body fluid extracted from a patient, is directly obtained from said patient, conducted through the extracorporeal device and, thereby, exposed to the molecules binding to the soluble cytokine receptor and is finally led back to said patient. This process may preferably be controlled manually and/or computer-assisted. In the device, the body fluid may be brought to or kept at any temperature sufficient for treating a body fluid, preferably the body fluid may be cooled to a temperature in the range of between 2°C and 5°C, between 5°C and 10°C, between 10°C and 15°C, between 15°C and 25°C, between 25°C and 37°C, or may be kept at body temperature, preferably at approximately 37°C. More preferably, the body fluid is cooled to a temperature in the range of between 2°C and 5°C or kept at body temperature, preferably at approximately 37°C, in particular kept at approximately 37°C. When returned into the patient, the body fluid may preferably bear body temperature, thus a temperature of approximately 37°C. The extracorporeal device may also serve as a combination of an offline and an online method. Then, e.g., the body fluid may be extracted from a patient in a pulsed mode, the soluble cytokine receptor may be removed in the extracorporeal device in a pulsed mode and/or the body fluid may be led back to the patient in a pulsed mode. Herein, the term "pulsed mode" means that there is no continuous flow, but the sample is rather treated batch-wise every few seconds or every few minutes, whereas the process comprising extracting the body fluid and removing the soluble cytokine receptor is conducted automatically.

Preferably, the extracorporeal device has been sterilized and/or endotoxin has been removed from the device. Sterilization may lead to a decreased concentration of pathogens and/or microorganisms such as, e.g., bacteria, viruses, fungals, moulds, bacterial spores, fungal spores, mould spores, worms, worm eggs, or eukaryotic protocoa. Sterilization may be accomplished by any means known in the art such as, e.g., employment of radiation (e.g., UV radiation, gamma radiation) or one or more antimicrobic agent(s) known in the art. An endotoxin may be any endotoxin known in the art, such as e.g., lipopolysaccharide (LPS) or delta endotoxin. Typically, an endotoxin originates from the cell wall of a gram- negative bacterium.

As used herein, the term "column" refers to any hollow chromatography device that is itself coated with a molecule binding to the soluble cytokine receptor and/or filled with a column matrix bearing a molecule binding to the soluble cytokine receptor. As used herein, the term "column matrix" refers to any chromatographic material and may preferably be a bead material. The beads may be spherical or may bear any other shape known in the art to conduct chromatographic material. The beads may be of any material known in the art to be useful for preparing chromatographic material such as, e.g., silica, sugar-based bead material (e.g., agarose, sepharose), plastic bead material (e.g., polystyrene). The beads may bear a neutral, a positive or a negative zeta potential. The column may be a flow-through device or may be used batch-wise. When using a flow-through method, the column may be, e.g, an ultrapheresis column, a high performance liquid chromatography (HPLC) column, a fast protein liquid chromatography (FPLC) column, a flash chromatography (flash) column, a Rapid Refluid Liquid Chromatography (RRLC) column, a Rapid Separation Liquid Chromatography (RSLC) column, an Ultra Fast Liquid Chromatography (UFLC) column, an Ultra Performance Liquid Chromatography (UPLC) column or any other chromatography column known by those skilled in the art. Chromatography is widely used in protein purification. When the molecule binding to the soluble cytokine receptor is immobilized on the surface of the column device and/or on the column matrix an affinity chromatographic device is obtained. However, this device may concomitantly separate molecules by size exclusion chromatography, hydrophobicity chromatography, etc. The chromatographic methods may be combined with one or more filter(s), membrane(s) and/or semi-permeable material(s).

The term "filter" as used herein may refer to any device bearing pores. The pores may have an average pore size of an average pore size in the range of few nanometers or even less for separating high-molecular weight molecules, such as. proteins, of different molecular weights from another or the pores may have an average pore size of few micrometers for separating solid particles such as cells (e.g., red blood cells (RBCs), leucocytes, tumor cells), from another and/or from the liquid compartments of a body fluid (e.g., blood or lymph). The filter may have a molecular exclusion size that excludes molecules of less than 1 ,000 Da, less than 10,000 Da, less than 25,000 Da, less than 50,000 Da, less than 75,000 Da, less than 100,000 Da less than 125,000 Da, less than 150,000 Da, less than 175,000 Da, less than 200,000 Da, less than 250,000 Da, or less than 300,000 Da. The filter may preferably be a capillary membrane filter with a pore size of between 0.001 and 10 μιη, more preferably of between 0.01 and 1 μηι, even more preferably of between 0.01 and 0.1 μπι, even more preferably of between 0.01 and 0.09 μηι, μηι, even more preferably of between 0.01 and 0.08 μιη, even more preferably of between 0.01 and 0.07, even more preferably of between 0.02 and 0.07 μιη, even more preferably of between 0.02 and 0.06, even more preferably of between 0.03 and 0.06 μπι, even more preferably of between 0.04 and 0.06 μηι, and most preferably of between 0.04 and 0.05 μηη. Alternatively, the a preferred filter may be a parallel plate filter with a pore size of between 0.001 and 10 μιη, more preferably of between 0.01 and 1 μιη, even more preferably of between 0.01 and 0.1 μηι, even more preferably of between 0.01 and 0.09 μπι, even more preferably of between 0.01 and 0.08 μπι, even more preferably of between 0.01 and 0.07 μηι, even more preferably of between 0.02 and 0.1 μηι, even more preferably of between 0.02 and 0.09 μηι, even more preferably of between 0.04 and 0.08 μηι, even more preferably of between 0.04 and 0.07 μηι, and most preferably of between 0.04 and 0.06 μπι. Several filters with the same, similar or different pore size(s) may be combined with another. The filter may be any kind of filter. The filter may be of any material, such as, e.g., plastic (e.g., nylon, polysterene), metal, alloy, glass, ceramics, cellophane, cellulose, or composite material. The filter may be hydrophobic or hydrophilic. The surface of the filter may be neutral or positively charged or negatively charged. The filter may be a part of a plasmapheresis and/or an ultrapheresis device as known in the art (see, e.g., WO 99/61085 and WO 01/37873). The filter may be a dead-end filter. Alternatively, the filter may also be a cross- flow filter. As used herein, the terms "cross-flow filter", "crossflow filter", "tangential flow filter" may be understood interchangeably. Filtration may be conducted batch-wise of in a continuous flow method. Preferably, the filtration is conducted in a continuous flow method. The filter may be combined with one or more column(s), membrane(s) and/or semi-permeable material(s).

The term "membrane" as used in the context of the solid support may be understood in the broadest sense as any thin, flexible solid material. The membrane may contain pores and serve as filter. Then at least part of the fluid flow goes through the membrane. Alternatively, the membrane may also be impermeable. Then, the fluid flow passes by the membrane on which molecules binding to the soluble cytokine receptor are immobilized. Several or even numerous membranes may be used in one device and form a functional array of membranes. The membrane may be a dialysis membrane. Then the membrane is permeable for some molecules (in particular small molecules) (semi-permeable). Dialysis is based on diffusion and osmosis, respectively, and is well known in the art. Dialysis is widely used in protein purification and is also used to provide an artificial replacement for lost kidney function in people with renal failure. The membrane may be of any material, such as, e.g., plastic (e.g., nylon, polysterene), metal, alloy, glass, ceramics, cellophane, cellulose, or composite material. The membrane may be hydrophobic or hydrophilic. The surface of the membrane may be neutral or positively charged or negatively charged. The membrane may be combined with one or more column(s), filter(s) and/or semi-permeable material(s).

The term "semi-permeable material" may refer to any material that allows the passage of some molecules whereas other molecules can not or nearly not pass. The semi- permeability can base, e.g., on size exclusion and/or on the charge of the semi-permeable material. The semi-permeable material may be a solid material. The semi-permeable material may be a membrane or a thick material and may be of any material, such as, e.g., plastic (e.g., nylon, polysterene), metal, alloy, glass, ceramics, cellophane, cellulose, or composite material. The semi-permeable material may be hydrophobic or hydrophilic. The surface of the semi-permeable material may be neutral or positively charged or negatively charged. The semi-permeable material may have a smooth or porous surface structure. The semi-permeable material may be combined with one or more column(s), filter(s) and/or membrane(s).

The term "bead" may refer to any small spherical particle. In particular a bead may be a microbead or a nanobead. A microbead typically bears an average diameter in the range of approximately from 1 μηι and 1000 μιη. A nanobead typically bears an average diameter in the range of approximately from 1 nm and 1000 nm. The bead may be of any material such as, e.g., of silica, of metal- or alloy-based material, of sugar-based material (e.g., agarose, sepharose), of plastic (e.g., polystyrene) or may be a quantumdot. The bead may be hydrophobic or hydrophilic. The surface of the bead may have a neutral, a positive or a negative zeta potential. The beads may be compact or may be porous. A bead may be used in a column or may be used in fluid. The beads may be combined with one or more column(s), filter(s), semi-permeable material(s) and/or membrane(s).

The term "tubing" may be understood as any hollow solid material through which a fluid may flow. It may be flexible or inflexible. A tubing may be column filed with a matrix, but may also be not filled with a matrix. Optionally, the tubing may be the tubing of a capillary electrophoresis (CE). The tubing may also be any kind of semi-permeable material. The tubing may be of any material, such as, e.g., plastic (e.g., nylon, polysterene), metal, alloy, glass, ceramics, cellophane, cellulose, or composite material and may be hydrophobic or hydrophilic. The surface of the tubing may be neutral, positively charged or negatively charged. A tubing may be combined with one or more column(s), filter(s), semi-permeable material(s) and/or membrane(s). The term "surface of a device" may refer to any kind of device. In particular, the device may be a therapeutic, a medical, or a laboratory device. The surface of the device may be of any material, such as, e.g., plastic (e.g., nylon, polysterene), metal, alloy, glass, ceramics, cellophane, cellulose, or composite material. The surface of the device may be hydrophobic or hydrophilic. The surface of the device may be neutral or positively charged or negatively charged.

As used herein, the term "implant" may be understood in the broadest sense as any device that is located or intended to be located inside the patients body (e.g., a subdermal implant). Typically, the implant thereby will get in contact with one or more body fluids, in particular extracellular matrix and/or blood. The implant may be a macroscopic device of few millimeters or even centimeters in size or may be a microscopic device such as a micro- or nanobead. The implant may be a therapeutically acceptable plastic material, a coated metal device (e.g., a titanium device) or may be a polysaccharide or polyacid that aggregates upon injection and thereby forms a depot (e.g., poly(lactic-co-glycolic acid) (PLGA)). The implant may or may not have a connection to the outside allowing to change the solid support the molecule binding to the at least one soluble cytokine receptors is immobilized on.

In a more preferred embodiment, the solid support forms part of an extracorporeal device further comprising at least one of the following:

(i) a primary filter to separate ultrafiltrate or plasma from the patient's blood;

(ii) a centrifuge to separate ultrafiltrate or plasma from the patient's blood;

(iii) a filter which removes components of a molecular weight of 120,000 Da or less from the blood, plasma or blood fraction;

(iv) a centrifuge which removes components of a molecular weight of 120,000 Da or less from the blood, plasma or blood fraction;

(v) means for administering radiation to patent's tissue; and/or

(vi) one or more cannula(s) and tubing(s) suitable for connecting the device to the patient.

As used herein, the term "primary filter" may be understood as a filter that is connected upstream to the solid support on which the soluble cytokine receptor-binding molecules are immobilized. The primary filter may protect said solid support from cells. When a primary filter is used, preferably only the flow-through of the primary filter, thus the ultrafiltrate, gets in contact with said solid support. As used herein, the term "ultrafiltrate" may be understood in the broadest sense as a solution, emulsion or suspension from which parts of the high- molecular weight components have been removed. Preferably, the ultrafiltrate is a solution from which at least the blood cells (e.g., red blood cells (RBCs) and lymphocytes) have been removed. Even more preferably, also the cellular fragments such as e.g., platelets have been removed. When cells and cellular fragments have been removed from blood, the person skilled in the art will recognize plasma. Optionally, further one or more coagulation factors may be removed. Then, the person skilled in the art will recognize serum. Optionally, in the ultrafiltrate, also protein fraction(s) above a given size exclusion level may be removed. The size exclusion level may be more than 1 ,000 Da, more than 10,000 Da, more than 25,000 Da, more than 50,000 Da, more than 75,000 Da, more than 100,000 Da more than 125,000 Da, more than 150,000 Da, more than 175,000 Da, more than 200,000 Da, more than 250,000 Da, or more than 300,000 Da. Preferably, molecules of a molecular weight of more than 120,000 Da may be removed from the ultrafiltrate.

The ultrafiltrate may be generated by any means known in the art. Exemplarily, it may be generated by means of a filter or by centrifugation. Centrifugation may be any centrifugation of liquids known in the art. Centrifugation may be conducted batch-wise of in a continuous flow method. Preferably, the centrifugation is conducted in a continuous flow method. Filtration and centrifugation may be combined with another, in particular in a continuous flow method, or may be combined with any other method known in the art. Optionally, centrifugation may be gradient centrifugation (e.g., sucrose gradient centrifugation, equilibrium centrifugation).

Before the body fluid is recirculated into the patient, the cells and/or protein faction(s) may optionally be reunited with the ultrafiltrate from which the soluble cytokine receptor has been removed. Preferably, at least most of the cells are added to the purified ultrafiltrate prior to being re- injected into the patient.

The term "administering radiation to patent's tissue" may comprise, but may not be limited to expose the patient, a patient's limb, a patient's organ, or a patient's tissue to any kind of radiation used for therapeutic purposes, such as, e.g., x-ray radiation, ultraviolet (UV) radiation (e.g., UV-A, UV-B, and/or UV-C radiation), alpha radiation, beta radiation, gamma radiation, or cosmic radiation. Radiation therapy may also include, but may not be limited to Intensity-Modulated Radiation Therapy (IMRT), 3-Dimensional Conformal Radiotherapy (3DCRT), Stereotactic body radiation therapy (SBRT), Stereotactic radiosurgery (SRS), image-guided radiation therapy (IGRT), Particle Therapy (e.g, proton therapy), Brachytherapy, Radioisotope Therapy (RIT) (e.g., with iodine- 131 , lutetium-177, strontium-89 and samarium ( ¹⁵³ Sm) lexidronam and/or yttrium-90). Preferably, a tumor and/or metastases of the patient are subjected to radiation.

As indicated above, the present invention refers to the sensitization of a tumor cell to a treatment with a chemotherapeutic agent. In this context, the chemotherapeutic agent may be any chemotherapeutic agent known in the art or a combination of two or more of the chemotherapeutics drugs in the arts.

Exemplarily, the chemotherapeutic agent may be selected from the group consisting of alkylating agents (e.g., melphalan, mechlorethamine, cyclophosphamide, chlorambucil, Ifosfamide), anti-metabolites (e.g., 5-fluorouracil, azathioprine, 6-mercaptopurine, mercaptopurine, pyrimidines, thioguanine, fludarabine, floxuridine, cytosine arabinoside (cytarabine), pemetrexed, raltitrexed, pralatrexate, methotrexate), plant alkaloids and terpenoids (e.g., vinca alkaloids (vincristine, vinblastine, vinorelbine, vindesine), taxanes (e.g., paclitaxel), Cytoxan), topoisomerase inhibitors (e.g., camptothecins: irinotecan, topotecan, etoposide, etoposide phosphate, teniposide), antineoplastica (e.g., doxorubicin (adriamycin), doxorubicin lipo, epirubicin, bleomycin)), actinomycin D, aminoglutethimide, amsacrine, anastrozole, antagonists of purine and pyrimidine bases, anthracyclines, aromatase inhibitors, asparaginase, antiestrogens, bexarotene, buserelin, busulfan, camptothecin derivatives, capecitabine, carmustine, a platin (e.g., cisplatin, carboplatin, oxaliplatin), cladribine, cytarabine, cytosine arabinoside, alkylating cytostatics, dacarbazine, daunorubicin, docetaxel, epirubicin, estramustine, etoposide, exemestane, fludarabine, fluorouracil, folic acid antagonists, formestane, gemcitabine, glucocorticoids, goserelin, hormones and hormone antagonists, hycamtin, hydroxyurea, idarubicin, irinotecan, letrozole, leuprorelin, lomustine, mercaptopurine, miltefosine, mitomycins, mitosis inhibitors, mitoxantrone, nimustine, procarbazine, tamoxifen, temozolomide, teniposide, testolactone, thiotepa, treosulfan, tretinoin, triptorelin, trofosfamide, cytostatically active antibiotics, everolimus, pimecrolimus, tacrolimus, azithromycin, spiramycin, sirolimus (rapamycin), roxithromycin, ascomycin, bafilomycin, erythromycin, midecamycin, josamycin, concancamycin, clarithromycin, troleandomycin, folimycin, tobramycin, mutamycin, dactinomycin, dactinomycin, rebeccamycin, a statin (e.g., cerivastatin, simvastatin, lovastatin, somatostatin, fluvastatin, nystatin, rosuvastatin, atorvasta ^' tin, pravastatin, pravastatin, pentostatin,), 4-hydroxyoxycyclophosphamide, bendamustine, thymosin a-1 , aclarubicin, fludarabine-5'-dihydrogen phosphate, hydroxycarbamide, aldesleukin, pegaspargase, cepharanthine, epothilone A and B, azathioprine, mycophenolate mofetil, c-myc antisense, b-myc antisense, betulinic acid, camptothecin, melanocyte stimulating hormone (a-MSH), activated protein C, IL- Ι β inhibitor, fumaric acid and esters thereof, dermicidin, calcipotriol, taclacitol, lapachol, β- lapachone, podophyllotoxin, betulin, podophyllic acid 2-ethyl hydrazide, sagramostim, (rhuGM-CSF), peginterferon a-2b, lenograstim (r-HuG-CSF), filgrastim, macrogol, cephalomannine, selectin (cytokine antagonist), CETP inhibitor, cadherins, cytokinin inhibitors, COX inhibitor (COX-2 or COX-3 inhibitor), angiopeptin, ciprofloxacin, fluroblastin, bFGF antagonists, probucol, prostaglandins, 1 , 1 l-dimethoxyeanthin-6-one, 1- hydroxy-1 l-methoxycanthin-6-one, scopoletin, colchicine, NO donors, pentaerythrityl tetranitrate, sydnonimines, S-nitroso derivatives, staurosporine, β-estradiol, a-estradiol, estriol, estrone, ethinyl estradiol, fosfestrol, medroxyprogesterone, estradiol cypionates, estradiot benzoates, tranilast, kamebakaurin, verapamil, ciclosporin A, paclitaxel and derivatives thereof such as 6- -hydroxy paclitaxel, baccatin, taxotere, mofebutazone, acemetacin, diclofenac, lonazolac, dapsone, o-carbamoyl-phenoxy-acetic acid, lidocaine, ketoprofen, mefenamic acid, piroxicam, meloxicam, chloroquine phosphate, penicillamine, hydroxychloroquine, auranofin, sodium aurothiomalate, oxaceprol, celecoxib, β-sitosterol, ademetionine, myrtecaine, polidocanol, nonivamide, levomenthol, benzocaine, aescin, elipticine, D-24851 (Calbiochem), colcemid, cytochalasin A-E, indanocine, nocodazole, bacitracin, vitronectin receptor antagonists, azelastine, free nucleic acids, nucleic acids incorporated into virus transmitters, DNA and RNA fragments, plasminogen activator inhibitor- 1 , plasminogen activator inhibitor-2, antisense oligonucleotide, VEGF inhibitors, IGF- 1 , active agents from the group of antibiotics such as cefadroxil, cefazolin, cefaclor, cefoxitin, gentamicin, penicillins, dicloxacillin, oxacillin, sulfonamides, metronidazole, antithrombotics, argatroban, aspirin, abciximab, synthetic antithrombin, bivalirudin, Coumadin, enoxaparin, GpIIb/IIIa platelet membrane receptor, antibodies to factor X _a inhibitor, heparin, hirudin, r-hirudin, PPACK, protamine, prourokinase, streptokinase, warfarin, urokinase, vasodilators, dipyramidole, trapidil, nitroprussides, PDGF antagonists, triazolopyrimidine, seramin, ACE inhibitors, captopril, cilazapril, lisinopril, enalapril, losartan, thioprotease inhibitors, prostacyclin,, vapiprost, interferon α, β and γ, histamine antagonists, serotonin blockers, apoptosis inhibitors, apoptosis regulators, NF-kB or Bcl- xL antisense oligonucleotides, halofuginone, nifedipine, tocopherol, molsidomine, tea polyphenols, epicatechin gallate, epigallocatechin gallate, boswellic acids and derivatives thereof, leflunomide, anakinra, etanercept, sulfasalazine, tetracycline, triamcinolone, procainimide, retinoic acid, quinidine, disopyramide, flecainide, propafenone, sotalol, amiodarone, natural and synthetically obtained steroids such as bryophyllin A, inotodiol, maquiroside A, mansonine, strebloside, hydrocortisone, betamethasone, dexamethasone, fenoprofen, ibuprofen, indomethacin, naproxen, phenylbutazone, acyclovir, ganciclovir, zidovudine, antimycotics, clotrimazole, flucytosine, griseofulvin, ketoconazole, miconazole, terbinafine, chloroquine, mefloquine, quinine, natural terpenoids, hippocaesculin, barringtogenol-C21-angelate 14-dehydroagrostistachin, agroskerin, agrostistachin, 17-hydroxyagrostistachin, ovatodiolids, 4,7-oxycycloanisomelic acid, baccharinoids B l , B2, B3 and B7, tubeimoside, bruceanol A, B and C, bruceantinoside C, yadanziosides N and P, isodeoxyelephantopin, tomenphantopin A and B, coronarin A, B, C and D, ursolic acid, hyptatic acid A, zeorin, iso-iridogermanal, maytenfoliol, effusantin

A, excisanin A and B, longikaurin B, sculponeatin C, kamebaunin, leukamenin A and B, 13, 18-dehydro-6-alpha-senecioyloxychaparrine, taxamairin A and B, regenilol, triptolide, cymarin, apocymarin, aristolochic acid, anopterin, hydroxyanopterin, anemonin, protoanemonin, berberine, cheliburin chloride, cicutoxin, sinococuline, combrestatin A and

B, cudraisoflavone A, curcumin, dihydronitidine, nitidine chloride, 12-beta- hydroxypregnadiene-3,20-dione bilobol, ginkgol, ginkgolic acid, helenalin, indicine, indicine-N-oxide, lasiocarpine, inotodiol, glycoside la, justicidin A and B, larreatin, malloterin, mallotochromanol, isobutyrylmallotochromanol, marchantin A, maytansine, lycoridicin, margetine, pancratistatin, liriodenine, bisparthenolidine, oxoushinsunine, aristolactam-AII, periplocoside A, ghalakinoside, deoxypsorospermin, psychorubin, ricin A, sanguinarine, manwu wheat acid, methylsorbifolin, chromones of spathelia, stizophyllin, akagerine, dihydrousambaraensine, hydroxyusambarine, strychnopentamine, strychnophylline, usambarine, usambarensine, daphnoretin, lariciresinol, methoxylariciresinol, syringaresinol, umbelliferone, afromoson, acetylvismione B, desacetylvismione A, vismione A and B), radiation therapy (e.g, Intensity-Modulated Radiation Therapy (IMRT), 3-Dimensional Conformal Radiotherapy (3DCRT), Stereotactic body radiation therapy (SBRT), Stereotactic radiosurgery (SRS), image- guided radiation therapy (IGRT), Particle Therapy (e.g, proton therapy), Brachytherapy, Radioisotope Therapy (RIT) (e.g., with iodine-13 1 , lutetium- 177, strontium-89 and samarium ( ^{, 5} Sm) lexidronam and/or yttrium-90)), antiangiogenic therapy (e.g., carboxyamidotriazole, TNP-470, CM101 , , Suramin, SU5416, Thrombospondin, VEGFR antagonists, angiostatic steroids + heparin, Cartilage-Derived Angiogenesis Inhibitory Factor, matrix metalloproteinase inhibitors, 2-methoxyestradiol, Tecogalan, tetrathiomolybdate, thalidomide, thrombospondin, soluble VEGFR- 1 and NRP- 1 , Angiopoietin 2, angiostatin (e.g., TSP-1 and TSP-2 angiostatin), endostatin, vasostatin, canstatin, calreticulin, platelet factor-4, TIMP and CDAI, Meth-1 and Meth-2, CXCL lOprothrombin (kringle domain-2), antithrombin III fragment prolactin, VEGI, SPARC, osteopontin, maspin, proliferin-related protein, restin ), kinase inhibitors (e.g., imatinib, imatinib mesylate, gefitinib, erlotinib, pazopanib, apatinib), polyclonal or monoclonal antibodies (e.g., rituximab, trastuzumab, cetuximab, bevacizumab, basiliximab, daclizumab), proteasome inhibitors (e.g., bortezomib), cytokine or hormone therapy (e.g., selective estrogen receptor modulator tamoxifen, or with IFN-a, IFN-β, IFN- γ, IL-4, IL-12, IL-18, platelet factor-4)), PARP inhibitors (e.g., iniparib, olaparib) and/or combinations of two or more thereof. In a preferred embodiment, the chemotherapeutic agent is an alkylating agent, preferably a nitrogen mustard alkylating agent, in particular Melphalan.

The person skilled in the art will know how to administer the particular chemotherapeutic agent to the patient. Depending on its chemical nature, it may be injected (e.g., subcutaneously (is.), intraperitoneal ly (i.p.) or intravenously ( .v.), intramusculary (i.m.), intraarterially (i.a.)), may be administered orally (e.g., via a pill, a capsule, a sirup, a juice), nasally (e.g., via a spray) or subdermally (e.g., via an ointment or a plaster).

It will be understood that the chemotherapeutic agent may also comprise one or more pharmaceutically acceptable carriers. A pharmaceutically acceptable carrier according the present invention may be any additive that is pharmaceutically acceptable, therefore, any additive that is non-toxic to the patient. Exemplarily, a pharmaceutically acceptable carrier may comprise a solvent such as, e.g., water, dimethyl sulfoxide (DMSO), ethanol, vegetable oil, paraffin oil or combinations thereof. Furthermore, a carrier may contain one or more detergents, one or more foaming agents (e.g., sodium lauryl sulfate (SLS)/ sodium doceyl sulfate (SDS)), one or more coloring agents (e.g., Ti0 ₂ , food coloring), one or more vitamins, one or more salts (e.g., sodium, potassium, calcium, zinc salts), one or more humectants (e.g., sorbitol, glycerol, mannitol, propylene glycol, polydextrose), one or more enzymes, one or more preserving agents (e.g., benzoic acid, methylparabene), one or more texturing agents (e.g., carboxymethyl cellulose (CMC), polyethylene glycol (PEG), sorbitol), one or more emulsifiers , one or more bulking agents, one or more glacing agents, one or more separating agents, one or more antioxidants, one or more herbal and plant extracts, one or more stabilizing agents, one or more polymers (e.g., hydroxypropyl methacrylamide (HPMA), polyethylene imine (PEI), carboxymethyl cellulose (CMC), polyethylene glycol (PEG)), one or more uptake mediators (e.g., polyethylene imine (PEI), dimethyl sulfoxide (DMSO), a cell-penetrating peptide (CPP), a protein transduction domain (PTD), an antimicrobial peptide, etc.) one or more antibodies, one or more sweeteners (e.g., sucrose, acesulfame K, saccharin Na, stevia), one or more counterstain dyes (e.g., fluorescein, fluorescein derivatives, Cy dyes, an Alexa Fluor dyes, S dyes, rhodamine, quantum dots, etc.), one or more homeopathic ingredients, one or more gustatory substances and/or one or more fragrances. The formulation may also be a retard formulation maintaining the blood level of the chemotherapeutic agent at a therapeutic level for a longer time such as few days or even a week or longer.

The molecule binding to at least one soluble cytokine receptor according to the present invention may be contacted with the body fluid of the patient at any time before, during or after the chemotherapeutic agent is administered to the patient for the treatment or prevention of a neoplasia, preferably cancer.

In a preferred embodiment, the molecule is contacted with the body fluid of the patient prior to, concomitantly with and/or subsequent to administering the chemotherapeutic agent to said patient.

When the molecule is contacted with the body fluid of the patient prior to administering the chemotherapeutic agent to said patient, it may be a pre-treatment for making the tumor cells in the patient more sensitive to the subsequently administered chemotherapeutic agent. In this context, preferably, the molecule is contacted with the body fluid of the patient not more than a month, more preferably not more than two weeks, even more preferably not more than one week, even more preferably not more than five days, even more preferably not more than four days, even more preferably not more than three days and most preferably not more than two days before the administration of the chemotherapeutic agent to said patient. The body fluid of the patient may also be contacted with the molecule according to the present invention few hours or even few minutes before the chemotherapeutic agent is administered to said patient.

When the molecule is contacted with the body fluid of the patient concomitantly with administering the chemotherapeutic agent to said patient, the body fluid of the patient may be contacted with the body fluid of the patient while the chemotherapeutic agent is administered.

When the molecule is contacted with the body fluid of the patient subsequent to administering the chemotherapeutic agent to said patient, it may be an after-treatment for making or maintaining the tumor cells in the patient more sensitive to the previously administered chemotherapeutic agent. In this context, preferably, the molecule is contacted with the body fluid of the patient not more than a month, more preferably not more than two weeks, even more preferably not more than one week, even more preferably not more than five days, even more preferably not more than four days, even more preferably not more than three days and most preferably not more than two days after the administration of the chemotherapeutic agent to said patient is terminated. The body fluid of the patient may also be contacted with the molecule according to the present invention few hours or even few minutes after the chemotherapeutic agent is administered to said patient.

Preferably, the contacting of the molecule binding to at least one soluble cytokine receptor may lower the concentration of said soluble cytokine receptor in the body fluid of the patient before, during and/or after the administration of the chemotherapeutic agent to the patient.

In a preferred embodiment, the concentration of the soluble cytokine receptor is lowered in the body fluid of the patient when the chemotherapeutic agent is administered to said patient.

As indicated above, this may preferably be achieved by a single or a repeated treatment previous to or concomitantly with the administration of the chemotherapeutic agent to the patient.

Herein, in a particularly preferred embodiment, the time interval between contacting said molecule with the body fluid of the patient and administering said chemotherapeutic agent is not longer than seven days, preferably not longer than 5 days, in particular not longer than 3 days.

It will be understood that the contacting of the body fluid of the patient and/or administering of the chemotherapeutic agent may be repeated two, three, four, five or even more often. Then the treatment is a repeated treatment.

In a preferred embodiment, the molecule is contacted with the body fluid of the patient repeatedly, preferably at least three times.

Preferably, by subjecting the body fluid of the patient, the concentration of the soluble cytokine receptor of interest is maintained at a lower level compared to the level observable without contacting the body fluid with the molecule according to the present invention for at least twelve h, at least 24 h, at least 48 h or even a week or longer.

Preferably, also the chemotherapeutic agent is administered to the patient repeatedly, preferably at least three times. Preferably, the concentration of the chemotherapeutic agent is maintained at a therapeutically active level for at least twelve h, at least 24 h, at least 48 h or even a week or longer. This may be achieved by repeated administration and/or by using a retard and/or depot administration method(s) well-known in the art.

Most preferably, the concentration of the soluble cytokine receptor of interest is maintained at a lower level compared to the level observable without contacting the body fluid with the molecule according to the present invention and, concomitantly, the concentration of the chemotherapeutic agent is maintained at a therapeutically active level for at least twelve h, at least 24 h, at least 48 h or even a week or longer.

As indicated before, the term "body fluid" may be understood in the broadest sense as any fluid obtainable from a body such as, e.g., blood, urine, cerebrospinal fluid, lymph, saliva and/or one or more secret(s) from any gland(s).

In a preferred embodiment, the body fluid is selected from the group consisting of blood, plasma, lymph and extracellular matrix.

Most preferably, the body fluid is blood, in particular blood in a patient who may bear one or more tumor cell(s) or who is at risk of having or developing one or more tumor cell(s).

As indicated above, the patient may have or may have no clinical symptoms of a neoplasia, in particular cancer.

In a preferred embodiment, the patient is at least partly resistant to the chemotherapeutic agent and/or is suffering from at least one undesired side effect caused by said chemotherapeutic agent.

As used herein, the term "partly resistant" may be understood in the broadest sense as a decrease in sensitivity to the chemotherapeutic agent. A partly resistant cell may require a chemotherapeutic agent concentration that is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 2foId, at least 3fold or even higher concentration to show the desired effect, i.e., for most chemotherapeutic agents to become apoptotic.

An undesired side effect may be, beside others, selected from the group consisting of hair loss, depression of the immune system, infections due to naturally occurring microorganisms in the patient's own gastrointestinal tract (including oral cavity) and skin anemia, cardiotoxicity (heart damage), hepatotoxicity (liver damage), nephrotoxicity (kidney damage), ototoxicity (damage to the inner ear), producing vertigo, encephalopathy (brain dysfunction), immunosuppression and myelosuppression, chemotherapy-induced nausea and vomiting (CINV), secondary neoplasm, infertility, teratogenic effects, neurological adverse effects, tumor lysis syndromered skin (erythema), dry skin, damaged fingernails, a dry mouth (xerostomia), water retention, and sexual impotence. Some medications can trigger allergic and pseudoallele reactions. As used herein, the terms "undesired side effect", "adverse effect" and "adverse side effect" may be understood interchangeably. As indicated above, the molecule binding to at least one soluble cytokine receptor according to the present invention may be used for the treatment or prevention of any neoplasia.

In a preferred embodiment, the molecule is for use in a method for treating cancer in the patient.

As used herein, the treatment of cancer may also include the prevention of a recurrence of cancer in a patient who has been subjected to an anticancer treatment before.

A further aspect of the present invention refers to a body fluid from which at least one soluble cytokine receptor has been removed for use in a method for sensitizing a tumor cell in a patient to a treatment with a chemotherapeutic agent.

In this context, the term "body fluid" as described above may also include any fluid obtainable from a body after one or more processing step(s) (e.g., centrifugation, filtration, cross filtration, precipitation and/or any other known method for fractioning biological fluids) such as, e.g., blood serum or blood plasma. As used herein, the terms "blood serum" and "serum" may be understood interchangeably. Likewise, the terms "blood plasma" and "plasma" may be understood interchangeably. Preferably, the body fluid is blood, blood plasma, blood serum or lymph.

Preferably, the body fluid may be obtained from the same patient who is subsequently treated therewith after at least one soluble cytokine receptor has been removed (autologous graft). Then, the obtained body fluid from which the soluble cytokine receptor has been removed may be re-injected or re-circulated into the patient the sample has been extracted from. Most preferably, the patient will retrieve his own blood (autologous blood) that is returned into his blood stream after being treated by means of the molecule and/or the method of the present invention.

Preferably, the body fluid is withdrawn from the patient, treated by means of the molecule and/or the method of the present invention and, subsequently, immediately conducted back into the patient's blood stream. However, the patient's autologous blood may also be stored before treatment and/or after treatment and conducting it back into the patient's blood stream.

Alternatively, the body fluid may be obtained from an individual and is infused into another patient (xenograft) or a mixture of autologous and heterologous body fluid(s). The body fluid may more preferably be used directly obtained from the patient or within few minutes or few days. When the samples are stored for more than few hours, the samples may preferably be stored at room temperature or in the fridge. As used in the context of the present invention, the terms "room temperature" and "ambient temperature" may be understood interchangeably. Alternatively, the samples may also be stored in a frozen, deep-frozen or freeze dried state and may then be stored at any temperature below the freezing point, such as, e.g., at -20°, -80°C or in liquid nitrogen. A freeze-dried powder may also be stored at ambient temperature. Obtaining the body fluid from a patient is typically of no health risk for the patient and can be also conducted by persons who are no medical experts such as, e.g., even the patient at home.

As used herein, the term "obtained from a patient" may be understood in the broadest sense as receiving a body fluid from the body of a patient by any means. Exemplarily, blood may be collected from a blood vessel, lymph may be corrected from a lymph vessel, cerebrospinal liquid may be obtained from the cerebrospinal lumen and/or urine and/or sweat may be collected. It will be understood that the body fluid may also be processed further. Exemplarily, blood plasma or blood serum may be extracted from the blood by any means known in the art. The body fluid may be used in any method of the present invention or may be stored at appropriate conditions for up to one hour, up to two hours, up to six hours, up to twelve hours, up to a day or even longer. A sample of body fluid stored for more than one day may be designated as a preserved sample.

As used herein, the term "preserved sample" may be understood in the broadest sense as any sample that has been stored for more than one day, more than two days, more than a week, more than two weeks, more than a month, more than two months, more than six months or more than a year.

The body fluid sample may be stored as the pure body fluid or may be stored as a sample further comprising other components of the sample as summarized in detail above. In general, all storage conditions suitable for the body fluid may be used. Exemplarily, the body fluid sample may be stored at room temperature, at 4°C, at -20°C, at -80°C or in liquid nitrogen. Therefore, the body fluid sample may be stored in a liquid or frozen state. Alternatively, the body fluid sample may be stored in a freeze-dried or dried state. The preserved sample may exemplarily be an autologous or heterologous blood preservation, a serum or plasma preservation, or a lymph preservation. Preferably, a blood preservation is an autologous blood preservation. Most preferably, the body fluid is blood that is obtained from a patient and is re-injected and re-circulated into said patient by any means known in the art. For instance, the sample may be injected by means of a tubing and/or an acus, a needle, a spicule or the like. These means are well-known in the context of dialyzers and kidney machines and can be used without particular expert skills.

As the present invention refers to the removal of the at least one soluble cytokine receptor from a body fluid, this removal may have an impact on the whole organism systemically, independent on where the tumor is located. Therefore, the effect is independent on barriers, such as the blood-brain barrier, the blood-testis barrier, the blood-milk barrier or the mother-embryo/fetus barrier. Therefore, the molecule of the present invention may also enable the systemic treatment of target tissue beyond the barrier. A patient suffering from a brain disease such as, e.g., e brain tumor (e.g., an astrocytome, or a glioblastome) may be treated by systemic treatment without targeting the brain directly. A fetus or embryo may be treated indirectly by treating the pregnant mother systemically. As used herein, the terms "systemic treatment" or "treating systemically" may be understood in the broadest sense as the treatment of a subject in total. Exemplarily, a body fluid such as, e.g., blood, may be extracted somewhere from the patient's body (e.g., from the basilica vein), conducted through the extracorporeal device of the present invention and, subsequently, being injected or recirculated somewhere in the patient's body (e.g., recirculated in the same or the other basilica vein), whereas the tissue to be treated (e.g., cancer tissue) may be located somewhere else in the patient's body (e.g., somewhere in the inner organs). Nevertheless, the treatment may be successful as a body fluid such as blood is circulating through the entire body of the patient. A treatment may be successful when the local concentration, but also when the overall concentration of the soluble cytokine receptor in the patient is depleted.

The therapy may also include the oral intake of an agent or a pharmaceutically acceptable composition thereof, the injection of an agent or a pharmaceutically acceptable composition thereof, the perfusion of an agent or a pharmaceutically acceptable composition thereof. Further, the removal of the soluble cytokine receptor may be combined with any surgical procedure known in the art such as, e.g., the section or resection of the tumor, the amputation of a limb, a breast, a testicle, or a certain tissue or parts thereof. Moreover, the removal of the soluble cytokine receptor may be combined with radiation therapy.

As already indicated above, in a preferred embodiment, the body fluid is selected from the group consisting of blood, plasma, lymph and extracellular matrix. More preferably, the body fluid is blood. Most preferably, the body fluid is autologous blood, i.e., blood obtained from a patient from which TNFR has been removed re-infused into the same patient. In this context, the soluble cytokine receptor may be removed from the body fluid by any means.

As used herein, "TNFR" without indicating may be sTNFR2 or sTNFRl .

In a preferred embodiment, the soluble cytokine receptor has been removed by means of a molecule according to the present invention as defined above.

All embodiments described above also apply to the following aspect referring to a method of the present invention.

In a further aspect, the present invention refers to chemotherapeutic agent for use in a method for treating a tumor in a patient, wherein at least one soluble cytokine receptor has been removed from a body fluid of said patient by means of a molecule according the present invention as defined above for sensitizing a tumor cell of said tumor in said patient to a treatment with said chemotherapeutic agent.

As indicated above, in a preferred embodiment, the patient is a cancer patient, preferably wherein said patient is at least partly resistant to the chemotherapeutic agent and/or is suffering from at least one undesired side effect caused by said chemotherapeutic agent.

In a further aspect, the present invention also relates to a method for sensitizing a tumor patient to a treatment with a chemotherapeutic agent, comprising the step of removing soluble TNF receptor 2 (sTNFR2) from the blood of said patient.

As described in Examples 1 and 2, the present inventors have found that by removing soluble TNF receptor 2 from the blood, the treated subject is sensitized for the treatment with a chemotherapeutic agent. Consequently, by removing sTNFR2 from the blood from the patient, it is possible to reduce the dose of the chemotherapeutic agent, which reflects an important improvement in tumor treatment.

The definitions and embodiments laid out in detail above in the context of the preceding aspects, i.e., in the context of the molecule, the body fluid and the chemotherapeutic agent according to the present invention, also apply to this method according to the present invention. Optionally, the method may be combined with the removal of one or more other plasma protein(s), preferably cytokines, chemokines, soluble cytokine receptors, other soluble decoy receptors, angiogenic factors, growth factors, and bone morphogenic factors more preferably molecules binding to sILRs, in particular molecules binding to sIL2R.

In a preferred embodiment, the method is for treating cancer in the patient, preferably further comprising administering a chemotherapeutic agent.

It will be understood that the patient may also be treated by a combination therapy comprising administering two or more chemotherapeutic agent(s).

As indicated above, in a preferred embodiment, the molecule according the present invention as defined above is contacted with the body fluid of the patient prior to, concomitantly with and/or subsequent to administering the chemotherapeutic agent to said patient.

As indicated above, the time interval between contacting said molecule with the body fluid of the patient and administering said chemotherapeutic agent is not longer than seven days, preferably not longer than 5 days, in particular not longer than 3 days.

As also indicated above, in a preferred embodiment, the molecule is contacted with the body fluid of the patient repeatedly, preferably at least three times.

Any of numerous chemotherapeutic drugs can be used in the methods or uses of the invention. These compounds fall into several different categories, including, for example, alkylating agents, antineoplastic antibiotics, antimetabolites, and natural source derivatives.

Examples of alkylating agents that can be used in the invention include busulfan, caroplatin, carmustine, chlorambucil, cisplatin, cyclophosphamide (i.e., Cytoxan), dacarbazine, ifosfamide, lomustine, mecholarethamine, melphalan, procarbazine, streptozocin, and thiotepa.

Examples of antineoplastic antibiotics include bleomycin, dactinomycin, daunorubicin, doxorubicin, idarubicin, mitomycin (e.g., mitomycin C), mitoxantrone, pentostatin, and plicamycin. Examples of antimetabolites include fluorodeoxyuridine, cladribine, cytarabine, floxuridine, fludarabine, flurouracil (e.g., 5-fluorouracil (5FU)), gemcitabine, hydroxyurea, mercaptopurine, methotrexate, and thioguanine.

Examples of natural source derivatives include docetaxel, etoposide, irinotecan, taxanes (e.g. paclitaxel), teniposide, topotecan, vinblastine, vincristine, vinorelbine, prednisone, and tamoxifen.

Additional examples of chemotherapeutic agents that can be used in the invention include asparaginase and mitotane.

Furthermore, also C2 ceramide can be used.

In an especially preferred embodiment, the chemotherapeutic drug is selected from the group consisting of actinomycin-D, mitomycin C, cisplatin, doxorubicin, etoposide, verapamil, podophyllotoxin, 5-FU, taxans such as paclitaxel, and carboplatin. sTNFR2 can be removed by any means known to the person skilled in the art. Preferably, sTNFR2 is removed with the help of a molecule binding to sTNFR2. Such molecules are discussed in detail above. Furthermore, such molecules also include molecules that do not bind specifically to sTNFR2, as e.g. polyclonal antibodies recognizing soluble TNF receptor 2 and soluble TNF receptor 1.

In a preferred embodiment, said molecule is an antibody against sTNFR2, preferably a monoclonal antibody against sTNFR2. Such antibodies have also been described above.

The invention further relates to a molecule binding to sTNR2 for use in a method for sensitizing a tumor patient to a treatment with a chemotherapeutic agent. Such molecules have been described in detail above, and all these embodiments also apply to this aspect of the invention.

In a preferred embodiment, the molecule is an antibody against sTNFR2, preferably a monoclonal antibody against sTNFR2.

The invention further relates to a method for treating a tumor in a patient, comprising administering to a patient a chemotherapeutic drug in an amount sufficient for the treatment of the tumor, wherein from the blood of said patient sTNFR2 has been removed. The treatment of tumors with chemotherapeutic agents is a standard procedure known to the person skilled in the art. All embodiments defined above also apply to this aspect of the invention. Examples of chemotherapeutic agents which can be used in the context of the present invention are given above.

Furthermore, the present invention also relates to a chemotherapeutic agent for use in a method for treating a tumor in a patient, wherein from the blood of said patient sTNFR2 has been removed. All embodiments discussed above also apply to this aspect of the invention.

The invention is further explained by the following example and figures, which are intended to illustrate, but not limit the scope of the present invention.

Figures

Figure 1 shows an exemplary treatment scheme.

Figure 2 shows the apheresis equipment. In the first part of the system (single arrow, — blood was pumped from the animal (A. carotis) via a plasma separator and retraced to the animal by a venous catheter. The separated plasma entered the second circuit (double- lined arrow, — and passed the adsorber before it was returned to the rat ( V. jugular is).

Figure 3 shows the mean tumor diameter in tumor-bearing Brown Norway rats treated with Sham apheresis (dashed line, open circles) or sTNFR2 apheresis (solid line, filled squares), respectively, and 0.25 mg/kg melphalan. The results of two independent study parts, study part I (3 A) and study part II (3B), are shown.

Figure 4 shows the change of mean tumor diameter during course of treatment of tumor- bearing Wag/Rij rats treated with Sham apheresis and 0.25 mg/kg low dose melphalan (Sham low, solid line, diamonds) or sT FR2 apheresis (sTNFR2 low, dotted line, squares), respectively Sham apheresis or sTNFR2 apheresis at high melphalan concentrations of 0.50 mg/kg (Sham high, dotted line, triangles, respectively sTNFR2 high, solid line, circles)

Figure 5 shows the body weight of Wag/Rij rats during the course of treatment with low doses of melphalan (0.25 mg/kg), Sham apheresis (Sham low, solid line, diamonds), sTNFR2 apheresis (sTNFR2 low, dotted line, squares) and with high doses of melphalan (0.50 mg/kg) and Sham apheresis (Sham high, dotted line, triangles), respectively sTNFR2 apheresis (sTNFR2 high, solid line, circles). Figure 6 shows the mean tumor diameter measured in tumor-bearing WAG/Rij rats during the course of treatment. CO-low and CO-high are control data where animals have been treated with "empty HiTrap columns" (containing 1 ml NHS activated Sepharose, that have undergone the same blocking procedure as antibody loaded columns), and repetitive administration of 0.25 mg/kg (low) respectively 0.50 mg/ml (high). Oncosorb-low are data where animals have been treated with apheresis using specifically loaded apheresis columns that can specifically bind sTNFR2, sTNFRl and IL-2R and repetitive administration of 0.25 mg/kg Melphalan.

Figure 7 shows the body weight of tumor-bearing WAG/Rij rats during the course of treatment. CO-low and CO-high are control data where animals have been treated with "empty HiTrap columns" (containing 1 ml NHS activated Sepharose, that have undergone the same blocking procedure as antibody loaded columns), and repetitive administration of 0.25 mg/kg (low) respectively 0.50 mg/ml (high). Oncosorb-low are data where animals have been treated with apheresis using specifically loaded apheresis columns that can specifically bind sTNFR2, sTNFRl and IL-2R and repetitive administration of 0.25 mg/kg Melphalan.

References

US 4,708,713

US 6,620,382

WO 01/37873

WO 05/107802

Brunstein F, Hoving S, Seynhaeve AL, van Tiel ST, Guetens G, de Bruijn EA, Eggermont AM, ten Hagen TL; Synergistic antitumor activity of histamine plus melphalan in isolated limb perfusion: preclinical studies; J Natl Cancer Inst. 2004 Nov 3;96(21): 1603-10.

Manusama ER, Nooijen PT, Stavast J, Durante NM, Marquet RL, Eggermont AM; Synergistic antitumour effect of recombinant human tumour necrosis factor alpha with melphalan in isolated limb perfusion in the rat; Br J Surg. 1996 Apr;83(4):551 -5.

Hoving S, Brunstein F, aan de Wiel-Ambagtsheer G, van Tiel ST, de Boeck G, de Bruijn EA, Eggermont AM, ten Hagen TL; Synergistic antitumor response of interleukin 2 with melphalan in isolated limb perfusion in soft tissue sarcoma-bearing rats; Cancer Res. 2005 May 15;65(10):4300-8. Lentz M, Kumar K; Reduction of plasma levels of soluble tumor necrosis factor and interleukin-2 receptors by means of a novel immunoadsorption column, Ther Apher Dial. 2008 Dec; 12(6):491 -9.

Examples

Example 1

Example 1 shows the influence of the removal of sTNFR2 in Wag/Rij rats bearing the immunogenic ROS-1 osteosarcoma on tumor growth and animal weight in the presence of systemically administered melphalan.

Aim

The project aimed to confirm the efficacy of the combination of Melphalan chemotherapy and the removal of sTNFR2 by apheresis in a tumor model using Wag/Rij rats bearing the immunogenic ROS-1 osteosarcoma. In addition, the experiments also aimed to find the optimal dosage of highly toxic Melphalan, combined with apheresis treatment.

1. Material and Methods

1.1. Animals

WAG/Rij rats with a mean body weight of 200 g (N = 20), were purchased from Harlan Laboratories (Niederlande) on 5 ^th of April 2012. As soon as the animals arrived in the test facility, they were housed individually in separated cages under following conditions:

• Temperature of 21+2°C

• Relative humidity (45-65%)

• 12-h/12-h dark/light cycle (lights on at 06:00 AM)

• Ad libitum access to tap-water and commercial pelleted laboratory chow (Art. No. VI 536; Ssniff® Spezialdiaten GmbH, D-59494 Soest, Germany)

During the study strict hygiene conditions were maintained. One to two weeks of acclimatization were allowed. During this period, the rats were periodically handled in order to limit animal stress during the study phase.

1.2. Preparation of Adsorber Columns

The preparation of columns was done by Biopheresis GmbH in Heidelberg. NHS activated 1 ml Sepharose columns were purchased from GE Healthcare (Order No. 17-0716.01). Based on the amount of antibodies used for human treatment using the OncoSorb® column, 0.12 mg of affinity purified goat anti -mouse sTNFR2 antibodies (purchased from R&D Systems Order No. AF-426-PB) were coupled according to the manufacturer's procedure to NHS activated Sepharose. The antibodies were able to remove secreted rat sTNFR2 molecules out of rat plasma as shown by affinity purification of the receptor from rat plasma. The antibodies of one type were from the same batch to ensure optimal comparability.

1.3. Tumor tissue handling and transfer:

Frozen ROS-1 tumor cells, delivered from Prof. T. Ten Hagen in March 2012 and stored at -70°C up to the culturing, were thawed on 3 ^rd of March and transferred in Minimum

Essential Medium Eagle (SIGMA- ALDRICH, M4655) containing 10% fetal calf serum (FCS) 2012. After cultivation in a culture flask with DMEM and 10% FCS during 8 days, the cells were adhered by trypsination and counted. 1.5 Mill, cells per animal were injected subcutaneously in tested Wag rij rats on 1 1 ^th of April.

Under light inhalation anaesthesia (Sevorane ^® ), ROS-1 tumor cells (1.500.000 in 500 μΐ per animal) were injected under the skin of the left flank region of the WAG rat to induce cancer growth (the method was detailed by Prof, ten Hagen; Rotterdam).

Rats with palpable tumors were instrumented with a chronic arterial and venous catheter. The catheters were inserted under general anaesthesia (i.p. injection of 0.8 mg Rompun ^® , Bayer and 4 mg Ketamin ^® , Sanofi, Germany) into the Arteria carotis and the Vena jugularis. Catheters were flushed with heparinized saline three times per week before the study.

When the tumor was grown to a mean diameter of >11 ± 3 mm, first chemotherapy and apheresis therapy was initiated on the following day.

1.4. Apheresis equipment

Before each apheresis treatment, a heparin bolus was given (90 IU/100 g body weight (b.w.)). The extracorporeal system was fully filled with heparinized saline and thereafter, the catheter endings were connected with the extracorporeal system as shown in Figure 2.

In the first part of the system, blood was pumped (multi-channel pump, Petro Gas Ausriistungen, Berlin, Germany) from the animal (A. carotis) via a plasma separator (Saxonia medical and Alpha Plan, Radeberg, Germany) and retraced to the animal by a venous catheter. The separated plasma entered the second circuit and passed the adsorber before it was returned to the rat (V. jugularis).

The following parameters were measured before and after each apheresis:

1. Bound sTNFR2 was determined by elution of the respective (bound) protein from the column after each apheresis by analysis of the eluate. 2. The following parameters were determined before and after each plasmapheresis: a. ) Hematocrit

b. ) White blood cell counts

c. ) Body temperature (one hour after the end of apheresis)

d. ) Body weight

e. ) Plasma was sampled (300 μΐ blood) for later determination of concentrations of TNF-a and IL-2.

The rats were sacrificed 21 days after first apheresis treatment. 50 % of the tumor (maximally a piece of 2 x 2 cm) was fixed in formalin to determine necrotic tissue and a comparable piece was shock-frozen and stored at -70°C for later histological investigation.

1.5. Preparation of the columns before and after apheresis:

Before each plasmapheresis each column was flushed with at least 6 ml 0.9% saline. After the apheresis treatment, each column was treated in the following way:

1. It was rinsed with 12 ml 0.9% saline.

2. It was washed with 3 ml glycine buffer (pH 2.8) to elute the bound proteins/factors.

3. It was washed with 6 ml 0.9% saline.

4. Column was kept in PBS-azide (for storage).

The eluate from each column was frozen for further analyses.

1.6. Chemotherapy and Apheresis:

The apheresis treatment was combined with intra-venous chemotherapy directly after the apheresis treatment . All animals were repeatedly administered a chemotherapeutic drug (Melphalan: Alkeran ^® , GlaxoSmithKline).

Each animal was treated with up to 6 apheresis cycles in intervals of 2-3 days (2-3 cycles per week). The duration of each plasmapheresis was one hour.

The adsorbers for the plasmapheresis procedure with a volume of 1 ml were provided by BioPheresis GmbH.

1.7. Treatment scheme

Table 1: Treatment scheme The first apheresis therapy was initiated directly when the tumor was grown to a mean diameter of 11±3 mm, followed by application of melphalan chemotherapy (day 0). On day 2, the second apheresis was performed and also the second Melphalan injection was administered. Melphalan was administered two times per week, for two weeks (four injections). Apheresis treatments were performed three times per week, for two weeks.

The animals were monitored without further treatment up to day 21.

Groups:

Sham: Sarcoma wearing rats with Sham apheresis (using the "empty HiTrap column") and 0.25 mg/kg low dose Melphalan (n=2) respectively 0.50 mg/kg (high dose) Melphalan (n=2)

sTNFR2 Sarcoma wearing rats with TNFR2 apheresis and low dose (0.25 mg/kg)

Melphalan (n=2) respectively 0.50 mg kg (high dose) Melphalan (n=2)

1.8. Monitored parameters:

Parameters monitored daily

• Body weight daily

• Health status

• Tumor growth, daily measurement of tumor size with a calliper

Parameters monitored before and after apheresis:

• Hematocrit

• Body temperature

At the end of the study, 21 days after tumor tissue transplantation, the tumor tissue was taken out, weighted and conserved for optional later histological investigation.

2. Ethics

The study was be performed under the animal license of the Institut fur Diabetes "Gerhard Katsch" GmbH , Karlsburg, Germany. Procedures related to this study have been reviewed and approved for the Institut fur Diabetes "Gerhard Katsch" GmbH, Karlsburg, Germany by the Animal Ethics Committee of the Country Mecklenburg- Vorpommern, Schwerin,

3. Data handling and mathematical analysis

All data were documented for statistical evaluations with Excel 2003. For calculation of the tumor size, the mean tumor diameter was calculated each day for each animal. Results were expressed as the means ± SD for each experimental group. By separate analysis the tumor diameter for each study part and the comparable tendency concerning the tumor growth depression was shown. The body weight and the tumor diameter were analyzed in dependence of the days after the first apheresis.

4. Results

4.1. Follow-up parameter

Tumour growth (delta mean tumour diameter)

The animals met inclusion criteria (mean diameter 1 1 ± 3 mm) 26 to 29 days after tumor cell injection. In the group of Sham apheresis / melphalan treated rats tumour size increased continuously during the twenty-one days of monitoring. In the sTNFR2 / melphalan group a tendency to a reduced tumour growth was shown from day 5 on.

The described effects are summarized in Table 2 and shown in figure 4 as the change of tumor size compared to initial size (delta mean diameter).

Sham low and Sham high are control data where animals have been treated with "empty HiTrap columns" (containing 1 ml NHS activated Sepharose, that have undergone the same blocking procedure as antibody loaded columns), and repetitive administration of 0.25 mg/kg (low) respectively 0.50 mg/ml (high). sTNFR2 high and low, respectively, are data where animals have been treated with apheresis using specifically loaded apheresis columns that can specifically bind sTNFR2.

The data demonstrate that removal of sTNFR2 by apheresis had a significant impact to reduce tumor growth even in the animals that were treated with the lower dose of 0.25 mg/kg Melphalan. The reduction of tumor growth was comparable to that observed with control animals that were treated with a higher dose of 0.50 mg/kg melphalan and that had been treated with a Sham apheresis that does not remove sTNFR2.

It appears that removal of sTNFR2 is sensitizing the tumor for melphalan treatment, which would allow a reduction of melphalan level in the plasma, which is beneficial for better toleration of the cytotoxic substance melphalan. Sham low day

Animal-No. 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

S 47 0.00 1.04 1.67 1.73 1.17 2.23 2.87 2.33 2.11 2.56 3.74 3.43 1.90 6.39 6.67 6.80 7.93 7.05 8.57 8.99 8.87 14,21

S 30 0.00 -0.62 0.39 0.30 1.28 0.74 2.01 1.98 3.40 3.47 2.83 4.93 4.57 5.82 5.22 7.20 7.12 7.44 9.68 7.55 8.66 mean 0.00 0.21 1.03 1.01 1.23 1.49 2.44 2.15 2.75 3.01 3.29 4.18 3.23 6.10 5.94 7.00 7.52 7.24 9.12 8.27 8.77

SD 0.00 1.17 0.91 1.01 0.08 1.05 0.61 0.25 0.91 0.65 0.64 1.06 1.88 0.41 1.03 0.28 0.57 0.27 0.78 1.02 0.15

STNFR2 low day

Animal-No. 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

S 40 0.00 1.34 2.53 0.98 0.21 2.48 1.14 2.52 1.10 3.44 3.50 4.16 3.62 4.05 4.99 5.32 6.14 6.24 7.09 6.34 8.85 8,42

S 48 0.00 -0.55 0.27 -1.00 0.58 0.73 0.92 1.11 0.78 1.60 -2.07 1.81 2.16 3.50 3.34 3.25 2.34 3.80 4.26 5.55 4.95 mean 0.00 0.39 1.40 -0.01 0.40 1.61 1.03 1.82 0.94 2.52 0.72 2.98 2.89 3.78 4.17 4.28 4.24 5.02 5.67 5.95 6.90

SD 0.00 1.33 1.60 1.41 0.26 1.24 0.16 1.00 0.23 1.30 3.94 1.66 1.03 0.39 1.17 1.47 2.69 1.73 2.01 0.56 2.76

Sham high day

Animal-No. 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

S 39 0.00 0.10 1.48 0.33 -0.26 0.61 1.88 2.12 1.60 2.67 1.82 4.50 3.74 4.47 6.62 4.79 5.75 6.63 6.26 5.82 7.84 7,14

S 42 0.00 -1.55 -1.60 -0.20 2.04 0.66 1.25 0.38 -0.33 0.51 1.73 2.77 0.09 1.50 1.47 1.64 2.57 1.94 0.42 5.24 3.01 mean 0.00 -0.72 -0.06 0.06 0.89 0.64 1.56 1.25 0.63 1.59 1.78 3.63 1.92 2.98 4.04 3.21 4.16 4.28 3.34 5.53 5.42

SD 0.00 1.16 2.17 0.38 1.62 0.03 0.44 1.23 1.36 1.52 0.06 1.23 2.58 2.10 3.64 2.22 2.25 3.32 4.13 0.41 3.41

STNFR2 high day

Animal-No. 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

S 21 0.00 -0.36 0.26 -0.23 0.72 0.85 -0.58 1.68 1.89 1.37 1.68 2.91 3.12 3.76 3.33 3.20 4.70 4.69 4.91 5.64 6.79 6,97

S 45 0.00 0.68 -0.67 0.38 0.78 1.29 0.94 0.48 1.94 3.79 2.72 2.40 3.44 2.75 2.77 3.46 3.16 3.30 6.29 5.91

mean 0.00 0.16 -0.21 0.08 0.75 1.07 0.18 1.08 1.92 2.58 2.20 2.66 3.28 3.25 3.05 3.33 3.93 3.99 5.60 5.78

SD 0.00 0.74 0.66 0.43 0.04 0.31 1.08 0.85 0.04 1.71 0.73 0.36 0.22 0.71 0.39 0.19 1.09 0.98 0.97 0.19

Table 2. Delta mean tumor diameter, the difference to tumor diameter measured at each animal at the days of the first apheresis, in mm. Sham low and Sham high are control data where animals have been treated with "empty HiTrap columns" (containing 1 ml NHS activated Sepharose, that have undergone the same blocking procedure as antibody loaded columns), and repetitive administration of 0.25 mg/kg (low) respectively 0.50 mg/ml (high). sTNFR2 high and low, respectively, are data where animals have been treated with apheresis using specifically loaded apheresis columns that can specifically bind sTNFR2.

Body weight

Two days after tumour tissue transfer, the rats had a body weight of 242 ± 8 g. The procedure of catheter insertion was followed by a decline of body weight during the next days and the initial body weight was reached again after 4 to 7 days. The start of apheresis treatments and Melphalan applications was followed by a decline in body weight gain surprisingly only in the Sham apheresis control group. The interruption of Melphalan treatment from day 2 to 7 followed by a slight reincrease of body weight in the both groups and the treatment with chemotherapeutic on day 7 induced a slight reduction again.

In the groups treated with TNFR-2 apheresis no prominent decline of body weight was induced by the Melphalan treatment.

It appears that removal of TNFR-2 by apheresis has a similar effect in a rat model as the administration of TNF in limb perfusion experiments, as published by Prof. Timo ten Hagen. He has shown that the Melphalan uptake in tumor tissue is enhanced after addition of TNF. This could lead to a reduction of melphalan level in the plasma, which is beneficial for better toleration of the cytotoxic substance Melphalan.

Body weight development of WG rats injected with sarcoma cells and treated with apheresis and Melphalan from day 0 on are summarized in Table 3 and shown in figure 5, where means of two animals per group are shown.

Sham low and Sham high are control data where animals have been treated with "empty HiTrap columns" (containing 1 ml NHS activated Sepharose, that have undergone the same blocking procedure as antibody loaded columns), and repetitive administration of 0.25 mg/kg (low) respectively 0.50 mg/ml (high). sTNFR2 high and low, respectively, are data where animals have been treated with apheresis using specifically loaded apheresis columns that can specifically bind sTNFR2.

Sham low day

Animal-No. 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

S 47 269 266 272 264 265 269 272 276 269 275 268 279 268 273 274 279 284 282 285 290 293 292

S 30 264 256 256 263 264 264 262 268 265 269 257 266 263 268 271 275 277 282 283 280

mean 267 261 264 264 265 267 267 272 267 272 263 273 266 271 273 277 281 282 284 285

SD 4 7 11 1 1 4 7 6 3 4 8 9 4 4 2 3 5 0 1 7

STNFR2

low day

Animal-No. 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

S 40 283 278 282 278 284 275 282 283 281 288 280 286 282 286 290 294 295 301 304 306 308

S 48 266 263 270 267 274 273 276 282 280 286 282 287 278 288 290 292 295 299 301 303

mean 275 271 276 273 279 274 279 283 281 287 281 287 280 287 290 293 295 300 303 305

SD 12 11 8 8 7 1 4 1 1 1 1 1 3 1 0 1 0 1 2 2

Sham high day

Animal-No. 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

S 39 275 266 269 261 268 268 272 270 276 268 266 267 269 275 280 280 282 283 287 287

S 42 266 252 243 243 245 250 249 254 251 255 252 259 254 263 268 269 272 278 283 281 mean 271 259 256 252 257 259 261 254 261 266 260 263 261 266 272 275 276 280 283 284

SD 6 10 18 13 16 13 16 n.a. 13 15 11 5 9 4 5 8 6 3 0 4

STNFR2

high day

Animal-No. 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

S 21 265 272 278 275 276 263 269 275 266 265 262 266 268 275 278 280 284 287 294 295 290

S 45 300 293 294 289 290 289 295 299 298 298 290 300 298 300 295 302 305 309 307

mean 283 283 286 282 283 276 282 287 282 282 276 283 283 288 287 291 295 298 301

SD 25 15 11 10 10 18 18 17 23 23 20 24 21 18 12 16 15 16 9

Table 3. Body weight (g) during the course of treatment. The first apheresis started on day 0. Sham low and Sham high are control data where animals have been treated with "empty HiTrap columns" (containing 1 ml NHS activated Sepharose, that have undergone the same blocking procedure as antibody loaded columns), and repetitive administration of 0.25 mg/kg (low) respectively 0.50 mg/ml (high). sTNFR2 high and low, respectively, are data where animals have been treated with apheresis using specifically loaded apheresis columns that can specifically bind sTNFR.2.

Hematocrit, Body temperature

No significant difference between the different groups has been observed during the observation period.

5. Conclusions

• Plasmapheresis was well tolerated in all animals.

• Removal of sTNFR2 by plasmapheresis in the presence of melphalan (0.25 mg/ kg rat body weight twice per week) has a tumor growth inhibiting effect.

• The data indicate that the tumor is sensitized for melphalan by removal of sTNFR2 with apheresis.

Example 2

Example 2 shows the influence of the removal of sTNFR2 simultaneous with sTNFR2 and IL-2R (Oncosorb® columns) in Wag/Rij rats bearing the immunogenic ROS-1 osteosarcoma on tumor growth and animal weight in the presence of systemically administered melphalan.

Aim

The project aimed to study if the efficacy of the combination of Oncosorb® Apheresis (the simultaneous removal of sTNFR2, sTNFRl and IL-2R) and Melphalan in a tumor model is comparable to the removal of sTNFR2 alone. In addition, the experiments also aimed to find the optimal dosage of low toxic Melphalan, combined with Oncosorb® apheresis treatment.

1. Material and Methods

1.1. Animals

WAG/Rij rats with a mean body weight of 200 g were purchased from Harlan Laboratories (Niederlande) on 5 ^th of April 2012. They were housed individually in separated cages under identical conditions as described for example 1. The rats were periodically handled in order to limit animal stress during the study phase.

1.2. Preparation of Adsorber Columns

The preparation of columns was done by Biopheresis GmbH in Heidelberg. The procedure, reagents and consumables were identical as described for example 2, except that instead of sTNFR2 antibodies alone, a combination of 3 antibodies equivalent to that used in Oncosorb® columns for use in humans was used: For the 1 ml "Oncosorb®" columns used in example 2 a mixture of 0.12 mg of affinity purified goat anti-mouse sTNFR2 antibodies (purchased from R&D Systems Order No. AF-426-PB), 0.12 mg of affinity purified goat anti-mouse sTNFRl antibodies (purchased from R&D Systems Order No. AF-425-PB) and 0.2 mg of affinity purified mouse anti-rat IL-2R ALPHA CHAIN antibodies (purchased from AbD SEROTEC Order No. MCA273EL) was immobilized on each 1 ml HiTrap Adsorber column. sTNFR2 antibodies were from the same batch as used in Example 1 to ensure optimal comparability.

1.3. Tumor tissue handling and transfer:

Tumor tissue handling and transfer was identical as described in Example 1.

1.4. Apheresis equipment

Apheresis equipment and procedure of apheresis treatment (including handling / rinsing of Adsorber columns before and after apheresis) was identical as for Example 1.

1.5. Chemotherapy and Apheresis:

The application of chemotherapy and the apheresis procedure was identical as for Example 1.

1.6. Treatment scheme

The first apheresis therapy was initiated directly when the tumor was grown to a mean diameter of 1 1±3 mm, followed by application of melphalan chemotherapy (day 0). On day 2, the second apheresis was performed and also the second Melphalan injection was administered. Melphalan was administered two times per week, for two weeks (four injections). Apheresis treatments were performed three times per week, for two weeks. The animals were monitored without further treatment up to day 21.

Table 4: Treatment scheme

Groups:

Control (CO) Sarcoma wearing rats with Sham apheresis (using the "empty HiTrap column") and 0.25 mg/kg low dose Melphalan (n=2) respectively 0.50 mg/kg (high dose) Melphalan (n=2)

Oncosorb® Sarcoma wearing rats with Oncosorb® apheresis and low dose (0.25

mg/kg) Melphalan medication (n=4) 1.7. Monitored parameters:

Parameters monitored daily

• Tumor growth (measurement of tumor size with a calliper)

• Body weight

Parameters monitored before and after apheresis:

• Hematocrit

• Body temperature

At the end of the study, 21 days after tumor tissue transplantation, the tumor tissue was taken out, weighted and conserved for optional later histological investigation.

2. Ethics

The study was be performed under the animal license of the Institut fur Diabetes "Gerhard Katsch" GmbH , Karlsburg, Germany. Procedures related to this study have been reviewed and approved for the Institut fur Diabetes "Gerhard Katsch" GmbH, Karlsburg, Germany by the Animal Ethics Committee of the Country Mecklenburg- Vorpommern, Schwerin,

3. Data handling and mathematical analysis

Data handling and mathematical analysis was identical as described for example 1 and 2.

4. Results

4.1. Follow-up parameter

Tumour growth (delta mean tumour diameter)

The animals met inclusion criteria (mean diameter 11 ± 3 mm) 26 to 29 days after tumor cell injection.

In the group of sham apheresis treated rats given low doses of Melphalan (CO-low), tumour size increased continuously during the twenty-one days of monitoring. The high dose Melphalan given on days 0, 2, 7 and 9, respectively, combined with sham apheresis treatment (CO-high) reduced tumour growth significantly during treatment.

Surprisingly, the tumor growth inhibiting effect of Oncosorb® apheresis in combination with low doses Melphalan treatment (Oncosorb®-low), was equally strong as that of high dose melphalan treatment and Sham apheresis (CO-high). A reduced tumour growth was observed both during treatment from day 2 to 9 (compared with CO-low) as well as after the end of the apheresis treatment and chemotherapeutic medication period from day 12 on. 4.2. Additional Findings

Several animals showed inflammatory reactions or reactions similar to an inflammatory reaction, i.e., fluid accumulation around the tumor. These reactions explain the outlier at day 10. For series conducted with Oncosorb® and a high dose of Melphalan, the aforementioned effect was more severe and partly led to outliers that are not shown.

Sham Apheresis + (4 x 0.25 mg/kg) low dose Melphalan

day after treatment start

Animal-No. 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

S 47 0.00 3.04 5.79 5.85 5.29 6.350 6.99 6.45 6.23 6.68 7.86 7.55 6.02 10.51 10.79 10.92 12.05 11.17 12.69 13.11 12.99 18.3

S 30 0.00 -0.61 0.39 0.30 1.28 0.75 2.01 1.98 3.39 3.47 2.83 4.93 4.57 5.81 5.22 7.20 7.12 7.44 9.68 7.55 8.65 10.7

S 35 0.00 0.21 1.67 3.39 4.20 5.29 4.29 3.84 5.89 6.09 6.16 6.67 9.69 8.58 9.25 10.03 11.43 9.55 13.61 14.59 13.2 mean 0.00 0.88 2.62 3.18 3.59 4.13 4.43 4.22 4.48 5.34 5.59 6.21 5.75 8.67 8.19 9.12 9.73 10.01 10.64 11.42 12.08 14.1

SD 0.00 1.91 2.82 2.78 2.07 2.98 2.49 3.16 1.52 1 .67 2.55 1.31 1.08 2.51 2.81 1.86 2.48 2.24 1.78 3.36 3.07 3.85

CO-high Sham Apheresis + (4 x 0.5 mg/kg) high dose Melphalan

day after treatment start

Animal-No. 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

S 39 0.00 0.10 1.48 0.33 -0.25 0.61 1.88 2.12 1.60 2.67 1.82 4.50 3.74 4.46 6.62 4.78 5.75 6.63 6.26 5.82 7.84 7.13

S 42 0.00 -1.55 -1.60 -0.20 2.04 0.65 1.25 0.38 -0.33 0.52 1.73 2.77 0.09 1.50 1.47 1.64 2.57 1.94 0.42 5.24 3.01 6.48 mean 0.00 -0.72 -0.06 0.06 0.89 0.63 1.56 1.25 0.63 1.59 1.78 3.63 1.91 2.98 4.05 3.21 4.16 4.28 3.34 5.53 5.42 6.80

SD 0.00 1.16 2.17 0.38 1.62 0.03 0.45 1 .23 1.36 1.52 0.06 1.23 2.59 2.09 3.64 2.22 2.25 3.32 4.13 0.41 3.41 0.46

Oncosorb- low Oncosorb Apheresis + (4 x 0.25 mg/kg) low dose Melphalan

day after treatment start

Animal-No. 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

S 29 0.00 1.14 1.48 0.59 3.08 2.01 2.99 2.70 3.38 3.09 14.10 11.82 9.33 8.58 8.31 7.01 8.84 8.61 7.84 8.11 8.86 9.37

S 28 0.00 -0.76 -1.63 -0.95 0.23 0.20 -0.59 0.03 0.50 -0.80 0.38 1.20 -0.83 1.08 2.89 2.07 4.94 4.42 3.10 3.66 4.49 4.02

S 22 0.00 1.04 0.94 1.35 2.09 2.30 2.51 0.93 2.65 4.35 4.52 5.89 4.45 4.07 4.66 4.95 7.10 7.78 10.51 9.90 3.33 13.0

mean 0.00 0.47 0.26 0.33 1.80 1.50 1.64 1.22 2.18 2.21 6.33 6.30 4.31 4.57 5.29 4.67 6.96 6.94 7.15 7.23 5.56 8.8

SD 0.00 1.07 1.66 1.17 1.45 1.13 1.94 1.36 1 .50 2.68 7.04 5.32 5.08 3.78 2.76 2.48 1.95 2.22 3.75 3.21 2.92 4.5

Table 5. Delta mean tumor diameter, the difference to tumor diameter measured at each animal at the days of the first apheresis, in mm. Sham low and Sham high are control data where animals have been treated with "empty HiTrap columns" (containing 1 ml NHS activated Sepharose, that have undergone the same blocking procedure as antibody loaded columns), and repetitive administration of 0.25 mg/kg (low) respectively 0.50 mg/ml (high). sTNFR2 high and low, respectively, are data where animals have been treated with apheresis using specifically loaded apheresis columns that can specifically bind sTNFR2.

Body weight

The body weight (cf., Figure 7) of the tested animals at the start of treatment was comparable between the investigated groups. Two days after tumor tissue transfer the rats had a weight of 242 ± 8 g. The procedure of catheter insertion followed a decline of body weight for several days. The initial body weight was reached again after 4 to 7 days.

The body weight of Melphalan/apheresis treated animals was followed by a decline in body weight gain in the both control groups (CO-low and CO-high). The weight loss was more prominent and with a longer duration in the group administered the higher Melphalan dose (CO-high; 0.5 mg/kg). The interruption of Melphalan treatment from day 2 to 7 was followed by a slight increase of body weight in the both groups. Repetitive treatment with chemotherapeutic agent on day 7 induced once more a slight body weight reduction.

In the both groups treated with Oncosorb® apheresis (Oncosorb® -low and Oncosorb® - high) nearly the same body weight behaviour during apheresis treatment was seen as in the control groups.

CO-low

day

Animal-No. 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

S 47 269 274 269 266 272 264 265 269 272 276 269 275 268 279 268 273 274 279 284 282 285 290 S 30 264 256 256 263 264 264 262 268 265 269 257 266 263 268 271 275 277 282 283 280 286 285 S 35 269 261 265 263 267 264 267 273 265 272 265 272 268 273 277 279 276 283 285 283 284 286 mean 267 265 263 265 268 264 264 269 269 273 263 271 266 274 270 274 276 281 284 281 286 288 SD 4 13 9 2 6 0 2 1 5 5 8 6 4 8 2 1 2 2 1 1 1 4

CO-high

day

Animal-No. 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

S 39 275 266 269 261 268 268 272 270 276 268 266 267 269 275 280 280 282 283 287 287 288

10 S 42 266 252 243 243 245 250 249 254 251 255 252 259 254 263 268 269 272 278 283 281 284 288 mean 271 259 256 252 257 259 261 254 261 266 260 263 261 266 272 275 276 280 283 284 286 288 SD 6 10 18 13 16 13 16 13 15 11 5 9 4 5 8 6 3 0 4 2 0

Oncosorb-low

day

Animal-No. 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 li

S 29 277 283 269 275 276 277 279 284 287 287 292 286 296 291 301 296 303 300 306 308 312 312 S 28 269 261 267 265 270 267 272 277 272 277 272 279 276 277 280 283 284 287 290 289 291 290 S 22 240 247 239 243 237 245 242 248 254 249 253 247 247 249 256 257 261 261 264 266 272 273 S 26 253 262 254 261 256 262 257 262 263 260 262 253 253 255 259 259 259 259 283 269 272 273 mean 260 263 257 261 260 263 263 268 269 268 270 266 268 268 274 274 277 277 286 283 287 287 SD 17 15 14 13 17 13 16 16 14 17 17 19 22 19 21 19 21 20 17 20 19 18

Table 6. Body weight (g) during the course of treatment. The first apheresis started on day 0. CO-low and CO-high are control data where animals ha been treated with "empty HiTrap columns" (containing 1 ml NHS activated Sepharose, that have undergone the same blocking procedure as antibody loaded columns), and repetitive administration of 0.25 mg/kg (low) respectively 0.50 mg/ml (high). Oncosorb-low are data where animals have been treated with apheresis using specifically loaded apheresis columns that can specifically bind sTNFR2, sTNFRl and IL-2R and repetitive administratio of 0.25 mg/kg Melphalan.

Hematocrit, Body temperature

No significant difference between the different groups has been observed during the observation period, indicating that the removal of sTNFR2 together with sTNFRl and IL- 2R by apheresis was well tolerated.

5. Conclusions

• Removal of sTNFR2 together with sTNFRl and IL-2R by Oncosorb® apheresis treatment in combination with low dose melphalan treatment (0.25 mg/ kg rat body weight twice per week) has a tumor growth inhibiting effect equivalent to high dose melphalan (0.50 mg/ kg rat body weight twice per week) and sham apheresis

• As a consequence, removing sTNFR2 together with sTNFRl and IL-2R by apheresis allows to reduce the concentration of Melphalan during a typical chemotherapy, thus reducing the strong side effects of highly toxic chemotherapy.

• Removal of sTNFR2, sTNFRl and IL-2R by Oncosorb® plasmapheresis in the presence of melphalan (0.25 mg/ kg rat body weight twice per week) has no effect on body weight loss, body temperature and Hematocrit compared to the control groups

• As in Example 1, this demonstrates that Plasmapheresis was well tolerated in all animals.