Tri-functional antibody purging of tumor cells in intraoperative autologous blood recovery

Abstract

The present invention relates to an extracorporeal method for removing tumor cells from intraoperatively collected blood, wherein trifunctional antibodies are used to bind leukocytes, tumor cells and Fc receptor positive cells. The invention also relates to the use of the above-mentioned extracorporeal method for removing tumor cells from intraoperatively collected blood, followed by reintroducing the purified blood or the red blood cell concentrate purified by this method into the patient from which the intraoperatively collected blood was obtained.

Description

TECHNICAL FIELD

[0001] The present invention relates to an in vitro method for removing tumor cells from intraoperative collected blood recovery using trifunctional antibodies, and the use of said in vitro method for removing tumor cells from intraoperative collected blood recovery, followed by reintroducing the purified blood or red cell concentrate purified by said method into the patient from which the intraoperative collected blood was obtained. BACKGROUND

[0002] Since the beginning of the AIDS epidemic in the early 1980s, there has been an increasing interest in alternatives to allogeneic blood transfusion, especially for elective surgery. In the United States and some European countries, more than 5% of blood donations are currently autologous, mainly obtained by preoperative donation. In addition to preoperative donation, intraoperative blood salvage (IBS) in the field of surgery (Table 1) is an important option to meet transfusion needs. During this IBS procedure, blood lost by a surgical patient is collected, washed, and can be used for reinfusion into the patient.

[0003] Briefly, blood flowing into the surgical field is sucked from this site into a specially designed container. Citrate or heparin anticoagulants are added, and the contents are centrifuged and / or filtered to remove leukocytes, clots, and debris. The IBS equipment used can range from simple, inexpensive, sterile bottles with anticoagulant to expensive, complex, high-speed cell washing devices (e.g. Medtronic Sequestra 1000, Cobe BRAT 2, Medtronic Autolog, Haemonetics Cell Saver 4+, etc.). The blood is then returned to the patient in a closed system. and Fresenius Bentzien et al., Anaesthesist 49:505, 2000; Serrit et al., J. Extra Corpor. Technol. 35(1):28, 2003; Carless et al., The Cochrane Review, In: The Cochrane Library, John Wiley & Sons, Ltd., issue 3 pp. 1-180, 2010). In the United States, nearly 1 million surgeries per year use the IBS procedure, which has become an integral part of hospital blood management and conservation programs.

[0004] Table 1. Benchmarks for blood salvage in cancer surgery

[0005] Effective blood preservation Blood resource relief Compatibility Blood supply Fast availability Optimal therapy for blood loss Cost efficiency Blood loss dependent applications No over transfusion At least after 1 L blood loss Transfusion deficit Improved efficacy of chemotherapy / radiation therapy High quality blood Fresh washed red blood cells Increased safety Reduced transfusion risks (e.g. infections, transfusion errors, difficult cross matching) No need for autologous pre-donation Costs of wasted units are more feasible for clinicians

[0006] In addition to the increased incidence of preoperative anemia, the preoperative donation of blood also poses a serious additional economic problem due to the high overall transfusion rate (Carless et al., Transfus. Med. 14:123, 2004). Moreover, in Italy, approximately 30% of preoperative autologous blood donations must be discarded because, according to regulatory guidelines, allogeneic transfusions do not include the unwanted autologous blood product per se. In this regard, IBS and subsequent autotransfusion are generally a safe and more cost-effective measure in blood management.

[0007] Advantages of intraoperative blood recovery

[0008] IBS is considered a safe and effective alternative to allogeneic red cell transfusion. Importantly, despite excellent viral diagnostics, the risk of transmitting HIV infection is 1 in 493,000 allogeneic transfusions, the risk of transmitting hepatitis C virus infection is 1 in 103,000, and the risk of transmitting hepatitis B virus infection is 1 in 63,000 (Schreiber et al., N. Engl. J. Med. 334:1685, 1996). The results of a study entitled "Serious Hazards of Transfusion" conducted from 1996 to 2001 documented these risks of allogeneic transfusion (Dzik et al., Transfusion 43:1190, 2003). Remarkably, the risk of transmitting infectious disease through contaminated transfusions appears to be small compared to the large risk of ABO incompatibility due to administrative or human error in the blood donation and transfusion process (Sazama, Transfusion 30:583, 1990; Dzik et al., Transfusion 43:1190, 2003). More than 70% of transfusion incidents can be attributed to incorrect transfusion components, primarily due to administration errors and errors in sampling, prescribing, and component collection (Dzik et al., Transfusion 43:1190, 2003). Preoperative and intraoperative autologous red cell transfusion bypasses these inherent risks of allogeneic transfusion altogether. In general, autologous blood recovery techniques have the advantage that no crystalloid or colloid needs to be infused to maintain blood volume (Table 1). With massive intraoperative blood loss, many liters of blood can be recovered, far exceeding other autologous techniques.

[0009] Suitability of patients for IBS

[0010] Intraoperative blood salvage has been in existence for over 25 years. It is widely used in cardiac surgery, vascular and trauma surgery, and liver transplantation. Contraindications for use are bacterial infection and the possibility of tumor cells being washed into the surgical field, as well as the use of microfibrillar collagen or other foreign bodies at the surgical site. Today, the use of IBS has also gained great interest in cancer surgery with high blood loss due to blood shortages from blood donors and fear of spreading infectious diseases. The reluctance of surgeons to use autotransfusion in cancer surgery has decreased as reports have found no increase in local recurrence or metastatic disease compared to standard survival data (Klimberg et al., Arch. Surg. 212:1326, 1986; Perseghin et al., Vox Sang. 72:221, 1997). As described by Vanderlinde et al. (BMJ 324:772, 2002), autotransfusion of red blood cells collected from preoperative blood donation can significantly reduce the rate of infection and recurrence during colorectal surgery compared to allogenic blood transfusion (Table 2). Some of these reviewed clinical trials are even more important as colorectal surgery itself is excluded from IBS recommendations due to the inherent risk of spreading bacterial infection.

[0011] Table 2 Clinical results from autologous versus allogenic blood transfusion randomized trials

[0012]

[0013] *Table excerpted from Vanderlinde et al., BMJ 324:772, 2002.

[0014] Nonetheless, one study clearly demonstrates that tumor cells can be detected in the surgical field, although the impact of tumor cells on cancer recurrence is unclear (Hansen et al., Arch. Surg. 130:387, 1995). For example, the number of tumor cells in the peripheral blood and IBS was compared in 61 patients with abdominal, orthopedic, urologic, gynecologic, or head and neck malignancies. Tumor cells were detected in the blood during tumor surgery in 57 of the 61 patients. These tumor cells were identified by proliferative capacity, invasiveness, and tumorigenicity, with a sensitivity of 10 tumor cells per 500 milliliters of blood (Hansen et al., Arch. Surg. 130:387, 1995). Interestingly, the number of tumor cells in the effluent blood was not related to the amount of blood loss, and circulating tumor cells were detected in the peripheral blood of only 26% of the patients. Therefore, it is estimated that the number of tumor cells in the blood that flows into the surgical field can be between 10 and 10 7between 0.1 and 1.0 x 10-6. Dale et al., Br. J. Surg. 75:581, 1988 and Muller et al., Anaesthesist 45:834, 1996, among others, have independently confirmed these results.

[0015] To further address the safety concerns of the risk of residual tumor cells in IBS samples, other methods need to be taken to effectively eliminate the contaminated tumor cells:

[0016] • One method used in conjunction with automated IBS devices (such as the Cell Saver®) is to additionally filter the sample through leukocyte reduction filters (such as the Pall RC400, RCEZ1T, RC XL-1) (Bontadini et al., Transfusion 34:531, 1994; Yaprak et al., Turk. J. Pediatr. 40:89, 1998; Gwak et al., Liver Transplant. 11:331, 2005). Through multiple trial evaluations, the safe transfusion of red blood cells collected by IBS does not compromise clinical outcomes (Edelman et al. Urology 47:179, 1996; Perseghin et al., Vox Sang. 72:221, 1996; Davis et al., BJU International 91:474, 2003).

[0017] • In another IBS method, the sample is irradiated at 50 Gy due to the fundamental principle of radiation sensitivity of nucleated cancer cells and the radiation resistance of non-nucleated red blood cells.

[0018] Due to the complex logistical requirements of this IBS / irradiation method, including additional personnel requirements, dosimetric issues, and the availability of suitable and certified irradiation equipment in the clinical department, the latter method is not conducive to widespread application. In contrast, the leukocyte-removing filtration procedure represents a preferred method to reduce residual tumor cells during IBS, although this technique still has the inherent risk that residual tumor cells can still pass through the filter.

[0019] Edmund A. M. Neugebauer: "Jahresbericht 2010, Institut fur Forschung in der Operativen Medizin", Witten / Herdecke, 1 March 2011 (2011-03-01), page 1-98 discloses trifunctional antibody Catumaxomab for elimination of free tumor cells from wound blood collected during surgery for autotransfusion during tumor surgery.

[0020] Alexandra Schoberth: "Entwicklung und Verbesserung von Methoden zur Charakterisierung disseminierter Tumorzellen bei soliden Tumoren", Dissertation an der Medizinischen der Ludwig-Maximilians- Universitat Munchen, 9 December 2004 (2004-12-09) discloses the use of trifunctional antibodies for ex vivo removal of tumor cells from autologous stem cell isolation products for reinfusion.

[0021] Schoberth A et al:,,A new class of trifunctional bispecific antibodies mediated efficient immunological purging of peripheral blood stem cells", European Journal of Cancer, vol. 37, 1 September 2001 (2001-09-01), page S51 describes that trifunctional bispecific antibodies can be used efficiently for immunological purging of peripheral blood stem cells.

[0022] EP 6317330 describes an ex vivo method for removing tumor cells from blood recovered during surgery using trifunctional bispecific antibodies that bind to T cells, tumor associated antigens on tumor cells and via their Fc part to Fc receptor positive cells. However, the binding to T cells, in particular to CD3 and CD28 as T cell surface markers, implies a certain risk of cytokine release and the cytokines can enter the collected red cell concentrate and be reinfused into the patient. Reinfusion of the red cell concentrate that can contain cytokines can cause a severe inflammatory reaction in the recipient. Furthermore, for patients with a reduced number of T cells, the method can not sufficiently remove tumor cells. SUMMARY

[0023] Problem to be solved by the invention

[0024] Therefore, in order to further improve the safety of removing residual tumor cells in cancer surgery, all known methods of reintroducing autologous blood, such as autologous blood obtained from the tumor patient wound during surgery, must be improved in order to reliably remove potentially contaminated tumor cells; the above-mentioned improvements should also include the efficient removal of tumor cells from the patient's autologous blood; in addition, the improvements should be easily implemented in devices such as Cell or Saver® or in any other method in order to reintroduce blood without or at least substantially without tumor cells into a patient undergoing surgery, wherein preferably the occurrence of the effects described is significantly reduced.

[0025] Summary of the invention

[0026] The new method discovered by the inventors is based on the formation of antibody-mediated multicellular complexes, i.e. conjugates and / or aggregates, comprising tumor cells recognized by antibody-driven tumor-associated antigens, and further recognition- leukocytes and / or Fc receptor-positive accessory cells and / or further tumor cells, followed by removal of the conjugates by centrifugation and / or filtration steps. Alternatively or in combination with the antibodies as claimed in claim 1, protein scaffolds that mimic antibodies can also be used.

[0027] Detailed description of the invention and preferred embodiments

[0028] In order to describe the present invention and to illustrate its preferred embodiments, the following discussion.

[0029] Unless otherwise defined, all technical and scientific terms used in this patent have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The singular terms "a," "an," and "the" include plural referents unless context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. The term "comprises" means "includes." All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including the

[0030] The present invention relates to:

[0031] (1) An in vitro method for removing tumor cells from blood recovery obtained intraoperatively, comprising the following steps:

[0032] - providing intraoperatively recovered blood which can contain tumor cells;

[0033] - contacting the intraoperatively recovered blood with

[0034] - selecting at least one antibody selected from the group consisting of bispecific, trispecific, tetraspecific and multispecific antibodies and bivalent, trivalent, tetravalent and multivalent antibodies, and / or

[0035] - at least one scaffold protein having a multispecific or multivalent binding property similar to antibodies, wherein the at least one antibody and / or the at least one scaffold protein comprises the following properties:

[0036] a) binding to a pan leukocyte antigen selected from the group consisting of at least one of CD11a, CD15, CD18, CD29, CD39, CD45, CD48, CD52, CD55, CD58, CD59, CD82, CD95, CD97, CD122, CD124, CD132 and CDw137;

[0037] b) binding to a tumor associated antigen on a tumor cell;

[0038] c) binding to Fc receptor positive cells via its Fc portion,

[0039] the at least one antibody and / or the at least one scaffold protein is capable of forming a three-dimensional network with at least tumor cells and leukocytes in the blood,

[0040] wherein the at least one antibody and / or the at least one scaffold protein is contacted with the intraoperatively recovered blood for 10 to 240 minutes, preferably 10 to 180 minutes, which is sufficient to crosslink tumor cells comprising the tumor associated antigen and leukocytes comprising the pan leukocyte antigen, and / or other tumor cells, to obtain a conjugate and / or aggregate comprising the antibody and / or the scaffold protein, wherein the tumor cells, the other tumor cells and the leukocytes can be present in the intraoperatively recovered blood, wherein the tumor cells are specifically recognized by the at least one antibody and / or the at least one scaffold protein; and

[0041] - mechanically removing the conjugate and / or aggregate from the intraoperatively recovered blood.

[0042] (2) Preferably, in the in vitro method according to (1), the at least one antibody is a trifunctional bispecific antibody.

[0043] (3) Preferably, in the in vitro method according to (2), the trifunctional bispecific antibody is selected from the group of antibodies having the following isotype combinations:

[0044] rat-lgG2b / mouse-lgG2a,

[0045] rat-lgG2b / mouse-lgG2b,

[0046] rat-lgG2b / human-lgG1,

[0047] mouse-[VH-CH1; VL-CL]-human-lgG1 / rat-[VH-CH1, VL-CL]-human-lgG1-[hinge]-human-lgG3*-[CH2-CH3]

[0048] [* = Caucasian allotype G3m(b+g) = does not bind to Protein A].

[0049] (4) Preferably, in the in vitro method according to any one of (1) to (3), the pan leukocyte antigen is CD52.

[0050] (5) Preferably, in the in vitro method according to any one of (1) to (4), at least two different antibodies are applied, whose tumor antigen specificity and / or pan leukocyte antigen specificity differ from each other.

[0051] (6) Preferably, in the in vitro method according to any one of (1) to (5), the conjugate consists of an antibody, a tumor cell and a leukocyte.

[0052] (7) Preferably, in the in vitro method according to any one of (1) to (6), the one or more antibodies and / or scaffold proteins are applied in an amount of 1 pg to 20 pg, preferably 1 to 10 pg or 1 to 5 pg or 1 to 2 pg per liter of intraoperatively recovered blood.

[0053] (8) Preferably, the in vitro method according to any one of (1) to (7) further comprises at least one of the following steps:

[0054] a) mixing the intraoperatively recovered blood with at least one anticoagulant prior to the addition of the at least one antibody and / or the at least one scaffold protein;

[0055] b) separating red blood cells from the conjugate and / or aggregate and further blood components by centrifugation, preferably by density gradient centrifugation;

[0056] c) optionally filtering the mixture to remove possibly remaining conjugate and / or aggregate and remaining cellular complexes; and

[0057] d) collecting the part containing red blood cells and further blood components in a separate container.

[0058] (9) Preferably, in the in vitro method according to any one of (1) to (8), the incubation time of the at least one antibody and / or the at least one scaffold protein with the intraoperatively recovered blood is between 10 and 90 minutes, further preferably between 20 and 60 minutes, optionally wherein the incubation is performed at a temperature between 19 and 25 °C, preferably at room temperature.

[0059] (10) Preferably, in the in vitro method according to any one of (1) to (9), the conjugates and / or aggregates are removed by centrifugation, filtration or a combination thereof.

[0060] (11) Preferably, in the in vitro method according to any one of (1) to (10), leukocytes and / or cell complexes containing tumor cells are removed in a separate step using a leukocyte adsorption / removal filter.

[0061] (12) Preferably, in the in vitro method according to one or more of (1) to (11), the tumor cells are from an epithelial, hematological or neuroectodermal tumor.

[0062] (13) Preferably, in the in vitro method according to one or more of (1) to (12), wherein a trifunctional bispecific antibody is additionally administered which binds to a tumor-associated antigen on the tumor cells and to an Fc receptor positive cell via its Fc part.

[0063] (14) Preferably, in the in vitro method according to any one of (1) to (13), the at least one antibody and / or the at least one scaffold protein comprises the property of binding to two different tumor-associated antigens. The two different tumor-associated antigens can be located on two different tumor cells, respectively, or can be located on the same tumor cell.

[0064] (15) Use of a bispecific, trispecific, tetraspecific or multispecific antibody, a bivalent, trivalent, tetravalent or multivalent antibody, and / or a scaffold protein having a multispecific or multivalent binding property similar to that of an antibody, in particular of a bispecific trifunctional antibody having the following properties:

[0065] a) binding to a pan-leukocyte antigen selected from the group consisting of at least one of CD11a, CD15, CD18, CD29, CD39, CD45, CD48, CD52, CD55, CD58, CD59, CD82, CD95, CD97, CD122, CD124, CD132 and cd137;

[0066] b) binding to a tumor-associated antigen on a tumor cell;

[0067] c) is capable of forming a three-dimensional network with tumor cells and leukocyte cells comprised in blood by binding to Fc receptor positive cells via its Fc part, in an in vitro method according to one or more of (1) to (13).

[0068] (16) Preferably, in the use according to claim 15, the at least one antibody and / or the at least one scaffold protein comprises the property of binding to two different tumor associated antigens. The two different tumor associated antigens can be located on two different tumor cells, respectively, or can be located on the same tumor cell.

[0069] (17) An antibody for use in the treatment of a tumor or cancer, wherein the antibody is selected from the group consisting of bispecific, trispecific, tetraspecific and multispecific antibodies, and bivalent, trivalent, tetravalent and multivalent antibodies, comprising the steps of:

[0070] - providing intraoperatively recovered blood which can contain tumor cells;

[0071] - contacting the intraoperatively recovered blood with the antibody, wherein the antibody comprises the properties of:

[0072] a) binding to a pan-leukocyte antigen selected from the group consisting of at least one of CD11a, CD15, CD18, CD29, CD39, CD45, CD48, CD52, CD55, CD58, CD59, CD82, CD95, CD97, CD122, CD124, CD132 and CDw137;

[0073] b) binding to a tumor associated antigen on a tumor cell;

[0074] c) binding to Fc receptor positive cells via its Fc part,

[0075] the antibody is capable of forming a three-dimensional network with at least tumor cells and leukocyte cells in blood,

[0076] wherein the antibody is contacted with the intraoperatively recovered blood for 10 to 240 minutes, preferably 10 to 180 minutes, which is sufficient to cross-link tumor cells comprising the tumor associated antigen and leukocyte cells comprising the pan-leukocyte antigen, and / or other tumor cells, to obtain a conjugate and / or aggregate comprising the antibody, wherein the tumor cells, the other tumor cells and the leukocyte cells can be present in the intraoperatively recovered blood and the tumor cells are specifically recognized by the antibody; and

[0077] - mechanically removing the conjugate and / or aggregate from the intraoperatively recovered blood.

[0078] (18) Preferably, the antibody for use in (17) comprises the property of binding to two different tumor-associated antigens. The two different tumor-associated antigens can be located on two different tumor cells, respectively, or can be located on the same tumor cell.

[0079] (19) A scaffold protein for use in the treatment of a tumor or cancer, wherein the scaffold protein has a multi-specific or multi-valent binding property similar to an antibody, comprising the steps of:

[0080] - providing intraoperatively recovered blood which can contain tumor cells;

[0081] - contacting the intraoperatively recovered blood with the scaffold protein, wherein the scaffold protein comprises the properties of:

[0082] a) binding to a pan-leukocyte antigen selected from the group consisting of at least one of CD11a, CD15, CD18, CD29, CD39, CD45, CD48, CD52, CD55, CD58, CD59, CD82, CD95, CD97, CD122, CD124, CD132 and CDw137;

[0083] b) binding to a tumor-associated antigen on a tumor cell;

[0084] c) binding to an Fc receptor positive cell via its Fc portion,

[0085] the scaffold protein is at least capable of forming a three-dimensional network with tumor cells and leukocytes in the blood,

[0086] wherein the scaffold protein is contacted with the intraoperatively recovered blood for 10-240 minutes, preferably 10-180 minutes, which is sufficient to cross-link tumor cells comprising the tumor-associated antigen and leukocytes comprising the pan-leukocyte antigen, and / or other tumor cells, to obtain a conjugate and / or aggregate comprising the scaffold protein, wherein the tumor cells, the other tumor cells and the leukocytes can be present in the intraoperatively recovered blood and the tumor cells are specifically recognized by the scaffold protein; and

[0087] - mechanically removing the conjugate and / or aggregate from the intraoperatively recovered blood.

[0088] (20) The scaffold protein for use according to (19) comprises the property of binding to two different tumor-associated antigens. The two different tumor-associated antigens can be located on two different tumor cells, respectively, or can be located on the same tumor cell.

[0089] Further preferred embodiments are described in the following description and examples in conjunction with the accompanying drawings. Further preferred features of the present application can be taken from the claims.

[0090] The presently described method is carried out in vitro, i.e. outside the human body. Intraoperative blood salvage (IBS) is well known in the art and is also described as "autologous blood salvage". Blood lost during surgery is salvaged and re-infused into the same patient from which the blood was lost during surgery.

[0091] The method of removing tumor cells from blood salvaged during surgery comprises the steps of the in vitro method described herein and, where appropriate, the content described in relation to the in vitro method applies accordingly to this method.

[0092] The presently described method is directed to the removal of tumor cells that can contaminate blood salvaged during surgery. The blood is preferably obtained from a patient who is treated with surgery and who has a tumor and / or cancer or is suspected to contain cells that can be considered as tumorigenic cells.

[0093] Preferably, the method is first of all by the surgeon to aspirate blood from the surgical area. The collection step is preferably carried out by means of an aspiration device and a diluent, such as sterofundin, can also be used during this step. Other diluents can also be used, such as 0.9% sodium chloride with heparin. The aspirated blood is then mixed with an anticoagulant to avoid coagulation of the blood. The aspirated blood can be collected in a reservoir until there is enough blood to be processed.

[0094] Preferably, the aspirated blood is diluted with a diluent, such as sterofundin and / or 0.9% sodium chloride with heparin.

[0095] "Anticoagulants" include anticoagulants of the class of anticoagulants, antiplatelet drugs, thrombolytic and fibrinolytic drugs, and metal ion chelators, such as citrate, acid citrate dextrose (ACD), EDTA, and oxalate, and the like. In a related aspect, anticoagulants include heparin and glycosaminoglycans, such as low molecular weight heparins, e.g. bemiparin, certoparin, dalteparin, enoxaparin, nadroparin, pamaparin, reviparin, and tinzaparin, and heparinoids, such as danaparoid, sulodexide, dermatan sulfate; direct thrombin (II) inhibitors, such as argatroban, bivalirudin, dabigatran, desirudin, hirudin, lepirudin, melagatran, semelagatran; Fact. Xa inhibitors (e.g. tick anticoagulant peptide), such as apixaban, otamixaban, rivaroxaban, and oligosaccharides, such as fondaparinux sodium and idraparinux sodium; vitamin K antagonists, such as acenocoumarol, clorindione, coumatetralyl, dicumarol, difenadione, ethyl biscoumacetate, phenprocoumon, fiendione, tioclomarol, and warfarin.

[0096] In one embodiment of the present application, the blood recovery thus collected can be tested in the presence of tumor cells, wherein it is particularly preferred to determine the type of tumor-associated antigens by methods known in the art, for example, by specifically reacting a labeled antibody with an epitope of a tumor-associated antigen to determine the type of tumor antigen to which the antibody binds and which is to be used in the present application.

[0097] In the present application, the term "tumor cell" refers to any cell that is capable of sustained division and forms a solid tumor or swarms abnormal cells in the blood. Preferably, in the present application, the tumor cells present in the intraoperatively recovered blood can be from an epithelial, hematological or neuroectodermal tumor.

[0098] Examples of tumors included in the present application, but not limited thereto, include sarcomas and carcinomas, including fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreatic cancer, breast cancer (including basal breast, ductal carcinoma and lobular carcinoma of the breast), lung cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, thyroid gland medullary carcinoma, thyroid gland papillary carcinoma, pheochromocytoma sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor, seminoma, bladder carcinoma, as well as tumors of the central nervous system (such as glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma and retinoblastoma). Further examples include epithelial tumors, hematological tumors and neuroectodermal tumors.

[0099] Preferably, in the method of the present application, the tumor cells are from an epithelial, hematological or neuroectodermal tumor.

[0100] In the present application, "immune cells" include leukocytes and Fc receptor positive cells. In the present application, "Fc receptor cells" refer to cells in which Fc receptors are present on the cell surface. Preferably, "Fc receptor positive cells" refer to one or more of monocytes, macrophages, dendritic cells, natural killer cells, neutrophils and eosinophils.

[0101] Preferably, the intraoperatively recovered blood is collected in a reservoir prior to contact with the antibody or the scaffold protein.

[0102] The intraoperative blood recovery is then contacted with an antibody or an appropriate protein scaffold that is capable of specifically binding to at least one epitope of at least one tumor-associated antigen of at least one tumor cell.

[0103] In the present application, "erythrocyte concentrate" means a concentrated erythrocyte.

[0104] In the present application, "cell aggregate" means cells adhering to each other, and the expression is interchangeable with "conjugate" or "cell conjugate". Likewise, "association" and "aggregation" are also interchangeable.

[0105] As a further requirement, the antibody must be capable of forming a three-dimensional network comprising at least the antibody and the tumor cells in the blood, so as to obtain an aggregate or conjugate comprising the antibody and tumor cells. In another particularly preferred embodiment of the present application, the antibody is also capable of binding to more than one tumor cell and / or immune cell, so as to form a three-dimensional network of antibody and tumor cells and optionally immune cells and optionally additional tumor cells. Since the composition, structure and size of the conjugate are caused by the formation of a multicellular complex, the conjugate can be removed from the IBS by centrifugation or filtration or a combination thereof. It will be appreciated that filtration and centrifugation are preferred methods, while the skilled person can recognize other methods for efficient removal of the conjugate comprising residual tumor cells.

[0106] Thus, the present application preferably focuses on the mechanical removal of conjugates and aggregates formed by binding and aggregation of antibodies with tumor cells and optionally immune cells and / or other tumor cells on tumor-associated antigens on the tumor cells, rather than depleting tumor cells carrying tumor-associated antigens by antibody-specific interaction and immune effector destruction of the tumor cells. When using antibodies as immunological reagents in a conventional manner in order to destroy tumor cells by interaction with immune cells, the present application preferably benefits from the ability of antibodies to cross-link antigens, in this case antigens located on tumor cells or fragments thereof. The effect of the present application is achieved by the interaction of antibodies with the tumor cells, in particular by removal of the three-dimensional network by centrifugation and / or filtration separation methods.

[0107] The present method can also be used in combination with other methods known in the art, in which antibodies or antibody-like molecules can be linked to magnetic components, such as magnetic beads or other structural elements, which facilitate the removal of antibody complexes by the structural elements, or the removal of tumor cells by cell sorting methods such as flow cytometry. Merely due to the formation of a three-dimensional network of antibodies with antigens, such as tumor-associated antigens, on tumor cells or fragments thereof, and optionally immune cells and / or other tumor cells, mechanical removal of the conjugate is possible. In this respect, in the present application, no other structural components such as magnetic beads or fluorescent molecules are required to facilitate the removal, which makes the present method simpler and easier to perform.

[0108] Within the limits of the invention, the invention is described and claimed for trifunctional bispecific antibodies and exemplified for trifunctional bispecific anti-CD52 x anti-EpCAM. Said trifunctional bispecific anti-CD52 x anti-EpCAM is directed against the tumor associated antigen EpCAM and binds via its Fc part to the pan-leukocyte marker CD52 and Fc receptor positive cells. The specific embodiments described in the examples have to be understood as exemplary embodiments which provide evidence for the feasibility of the invention. After providing evidence that catumaxab removes tumor cells from IBS very well, a proof of concept is given for the principle on which the presently claimed method is based. After providing this evidence, it will be inevitable for the skilled person to extend this concept to other tumors and other antibodies which are capable of interacting with said tumor cells and optionally said immune cells to provide a three-dimensional network, i.e. a multicellular complex which can be removed by centrifugation and / or filtration and the like.

[0109] Preferably, the antibodies of the present invention are specifically selected from trifunctional antibodies. Preferably, the trifunctional antibodies in the present invention can be bispecific, trispecific, tetraspecific and multispecific antibodies. The trifunctional antibodies described above refer to trifunctional bispecific antibodies. However, if trispecific antibodies, tetraspecific antibodies and multispecific antibodies also exhibit the same properties or effects, the trifunctional antibodies can also refer to trispecific antibodies, tetraspecific antibodies and multispecific antibodies as used herein.

[0110] The antibodies of the present invention are specifically selected from trifunctional antibodies. Bispecific antibodies are defined as antibodies which are capable of binding to two different types of antigens, preferably by their variable regions; trispecific antibodies are characterized by binding to three different types of antigens, preferably by their variable regions; tetraspecific antibodies are characterized by binding to four different types of antigens, preferably by their variable regions, and multispecific antibodies are defined as antibodies which are capable of binding to a plurality of different types of antigens, preferably by their variable regions. As a specific example, the trifunctional bispecific antibody anti-CD52 x anti-EpCAM is defined by binding to the tumor associated antigen EpCAM on the one hand and to the pan-leukocyte marker CD52 on the other hand and by binding to accessory cells via its Fc part.

[0111] The above described bispecific, trispecific, tetraspecific and multispecific antibodies can be monovalent, bivalent, trivalent, tetravalent or multivalent. Antibodies with monovalent binding properties are defined as antibodies that are capable of binding one tumor associated antigen. Bivalent monoclonal antibodies are antibodies that are capable of binding two tumor associated antigens or one tumor associated antigen and one immune cell associated antigen. Trivalent monoclonal antibodies are antibodies that are capable of binding three different tumor associated antigens or two tumor associated antigens and one immune cell associated antigen or one tumor associated antigen and two immune cell associated antigens. Tetravalent monoclonal antibodies are antibodies that are capable of binding four different tumor associated antigens or two different tumor associated antigens (two identical antigen binding arms per antigen) or two / three tumor associated antigens and one immune cell associated antigen or two tumor associated antigens and two immune cell associated antigens. Multivalent monoclonal antibodies are antibodies that are capable of binding one or more tumor associated antigens and / or one or more immune cell associated antigens. The term "binding to a tumor associated antigen" is defined as binding to an epitope of said tumor associated antigen on a tumor cell. The present invention only encompasses antibodies with a trifunctional bispecific format as described in claim 1. All other antibody descriptions are only for reference.

[0112] A general description of bifunctional or trifunctional antibodies is described by Kontermann RE (ed.), Springer Heidelberg Dordrecht London New York, pp. 1-28 (2011) which is of importance for the present patent application with bispecific or trispecific (trivalent, trivalent and tetravalent binding properties of trivalent) formats with binding properties to one tumor associated antigen and one or more leukocyte surface antigens (i.e. immune system cells).

[0113] · Bispecific antibody formats with bivalent antigen binding characteristics:

[0114] e.g. scFv (like BiT class), Db, scDb, dsDb, DART, dAb2 / VHH2, knob-in-hole derivative, SEED-IgG, hetero fc-scFv, Fab-scFv, CrossMabs

[0115] · Bis(tri)specific antibody formats with trivalent antigen binding characteristics:

[0116] e.g. triabody, DNL-F(ab)3, scFv 2-CH1 / CL , dAb3, Fab-scFv2, IgG-scFab

[0117] · Bis(tri)specific antibody formats with trivalent antigen binding characteristics:

[0118] e.g. IgG-scFv, scFv-IgG, scFv-Fc, F(ab')2-scFv2, sDb-Fc, scDb-C H 3, Db-Fc, scFv2-H / L, DVD-Ig, tandAb, scFv-dhlx-scFv, dAb2-IgG, two-in-one mAb, mAb 2 , dAb-IgG, dAb-Fc-dAb.

[0119] Further examples of antibodies to be used according to the present application are shown in Figure 2

[0120] Further antibodies to be used according to the present application are described in the following references:

[0121] Muller D and RE Kontermann. In: Bispecific Antibodies. Kontermann RE (ed.), Springer Heidelberg Dordrecht London New York, pp. 83-100 (2011)

[0122] scFv (BiTE)

[0123] Baeuerle PA, Zugmaier G and D Ruttinger. In: Bispecific Antibodies. Kontermann RE (ed.), Springer Heidelberg Dordrecht London New York, pp. 273-288 (2011)

[0124] DVD-Ig

[0125] Tarcsa E, Fraunhofer W, Ghayur T, Salfeld J and J Gu. In: Bispecific Antibodies. Kontermann RE (ed.), Springer Heidelberg Dordrecht London New York, pp. 171-186 (2011)

[0126] DNL-derivatives

[0127] ​Chang C-H, Rossi EA, Sharkey RM, DM Goldenberg. In: Bispecific Antibodies. Kontermann RE (ed.), Springer Heidelberg Dordrecht London New York, pp. 199-216 (2011)

[0128] Two-in-one antibodies

[0129] Koeing P and G Fuh. In: Bispecific Antibodies. Kontermann RE (ed.), Springer Heidelberg Dordrecht London New York, pp. 187-198 (2011)

[0130] CrossMabs

[0131] Schaefer et al. Proc. Natl. Acad. Sci. USA 108: 11187 (2011)

[0132] Preferably, two or more tri-functional antibodies with different specificities can be combined for mediating the formation of said conjugate.

[0133] The tri-functional bispecific antibodies used in the present application are directed against a pan leukocyte antigen selected from the group consisting of CD11a, CD15, CD18, CD29, CD39, CD45, CD48, CD52, CD55, CD58, CD59, CD82, CD95, CD97, CD122, CD124, CD132 and CDw137.

[0134] Preferably, the tri-functional bispecific antibodies used in the present application are directed against CD11a, CD18, CD45, CD52, CD82 or CD97. In another preferred embodiment, the antibodies are directed against CD52.

[0135] In a preferred embodiment, the antibodies used in the present application are monoclonal antibodies. This is especially true for the tri-functional bispecific antibodies described in detail herein.

[0136] Proteins with a relatively defined three-dimensional structure are often referred to as protein scaffolds. These protein scaffolds can be used as reagents for the design of artificial engineered antibodies. These scaffolds often contain one or more regions that are amenable to specific or random sequence variation, and often such sequence randomization is performed to generate a library of proteins from which a desired antibody scaffold can be selected. Such scaffolds are very useful in the field of antibody design.

[0137] These antibody scaffolds are non-immunoglobulin proteins that mimic the properties of monoclonal antibodies in binding activities to tumor cells and immune cells. The scaffolds often include loops or domains that form the binding side of the antibody scaffold. These antibody mimics can be used for the purpose of designing proteins that are capable of binding to virtually any compound of interest. This directed evolution approach can yield antibody-like molecules with high affinity to an antigen of interest. In addition, these scaffolds can be used to display defined exposed loops (e.g., loops previously selected at random for antigen binding) to guide the evolution of molecules that bind to such introduced loops. Methods for obtaining antibody-like scaffold proteins are known in the art. One possible method for obtaining antibody-like scaffold proteins is described below.

[0138] A first screening method for isolating or identifying a random or mutated protein of interest involves: (a) contacting a compound of interest with a candidate protein, said candidate protein being a derived non-antibody protein comprising a domain having an immunoglobulin-like fold, said non-antibody protein being derived from a reference protein by having a mutated amino acid sequence, wherein said non-antibody protein binds to a compound that is not tightly bound by the reference protein with a Kd of at least 1 μM, wherein said contacting is performed under conditions that allow for the formation of a compound-protein complex; and (b) obtaining from the complex the derived protein bound to the compound.

[0139] A second screening method is used to isolate or identify a compound that binds to a tumor-associated protein of interest. This method starts with a non-antibody protein that includes a domain having an immunoglobulin-like fold and has a mutated amino acid sequence by being derived from a reference protein, wherein the non-antibody protein binds to a compound that is not tightly bound by the reference protein with a Kd of at least 1 μM. The derived protein is then contacted with a candidate compound (a tumor-associated antigen or an epitope thereof), wherein the contacting is performed under conditions that allow for the formation of a compound-protein complex, and the compound bound to the derived protein is obtained from the complex. Again, this general technique can be used for any protein.

[0140] A further method for obtaining a non-antibody protein that binds to a compound of interest (a tumor-associated antigen or an epitope thereof) is described below. One such method involves: (a) providing a non-antibody scaffold protein comprising an immunoglobulin-like fold, wherein the scaffold protein does not bind to the compound with a Kd of tighter than 1 μM; (b) generating mutant derivatives of the non-antibody scaffold protein, thereby generating a library of mutant proteins; (c) contacting the library with the compound; (d) selecting at least one derivative protein from the library that binds to the compound with a Kd of at least 1 μM; (e) optionally repeating steps (b) - (d) using the product of the previous step (d) in place of the non-antibody scaffold protein in repeated step (b). Again, this general technique can be used for any protein.

[0141] The scaffold proteins thus prepared mimic the functions of the antibodies described above and below and can be used in place of or in combination with immunoglobulin-based antibodies. In the present application, the trifunctional antibodies can be replaced by the scaffold proteins as long as both display the same functions in binding leukocytes, in binding tumor-associated antigens on tumor cells and in binding Fc receptor positive cells via their Fc portion, to obtain blood containing cell aggregates for intraoperative recovery.

[0142] In the present application, the trifunctional antibodies and the scaffold proteins can be used individually or in combination.

[0143] The trifunctional bispecific and trispecific antibodies are described in more detail below.

[0144] Preferably, the antibodies used according to the present application comprise a binding site for Fcγ (Fcy) receptors type I, II and / or III in their Fc portion.

[0145] Preferably, the antibodies used according to the present application are capable of binding monocytes, macrophages, dendritic cells, natural killer cells, neutrophils and / or eosinophils as Fcy receptor positive cells.

[0146] In the present application, tumor-associated antigens mean antigenic substances produced in tumor cells.

[0147] Preferably, the tumor-associated antigens bound by the antibodies or the scaffold proteins, the trifunctional bispecific antibodies are selected from the group consisting of EpCAM, Her2neu, EGFR, CD30, CD20, CD22, MUC1, MUC1* with altered glycosylation pattern, PSMA, CD33, MCSP, cMet, EphA2, endosialin, carbonic anhydrase, IGF-1R, FAP alpha, CD19, GD2, CEA, FR, proteoglycan, G250, GC182, GT468, GT512.

[0148] More preferably, the tumor-associated antigen, antibody or scaffold protein, trifiinctional bispecific antibody of the present application binds to EpCAM, Her2 / neu, MUC1, EGFR, EphA2, GD2 or CD20.

[0149] Preferably, the trifunctional bispecific antibody of the application is directed against one pan-leukocyte antigen and one tumor-associated antigen, wherein the combination of the two antigens is selected from the group consisting of: EpCAM / CD11a, EpCAM / CD15, EpCAM / CD18, EpCAM / CD29, EpCAM / CD39, EpCAM / CD45, EpCAM / CD48, EpCAM / CD52, EpCAM / CD55, EpCAM / CD58, EpCAM / CD59, EpCAM / CD82, EpCAM / CD95, EpCAM / CD97, EpCAM / CD122, EpCAM / CD124, EpCAM / CD132, EpCAM / CDw137, Her2neu / CD11a, Her2neu / CD15, Her2neu / CD18, Her2neu / CD29, Her2neu / CD39, Her2neu / CD45, Her2neu / CD48, Her2neu / CD52, Her2neu / CD55, Her2neu / CD58, Her2neu / CD59, Her2neu / CD82, Her2neu / CD95, Her2neu / CD97, Her2neu / CD122, Her2neu / CD124, Her2neu / CD132, Her2neu / CDw137, EGFR / CD11a, EGFR / CD15, EGFR / CD18, EGFR / CD29, EGFR / CD39, EGFR / CD45, EGFR / CD48, EGFR / CD52, EGFR / CD55, EGFR / CD58, EGFR / CD59, EGFR / CD82, EGFR / CD95, EGFR / CD97, EGFR / CD122, EGFR / CD124, EGFR / CD132, EGFR / CDw137, CD30 / CD11a, CD30 / CD15, CD30 / CD18, CD30 / CD29, CD30 / CD39, CD30 / CD45, CD30 / CD48, CD30 / CD52, CD30 / CD55, CD30 / CD58, CD30 / CD59, CD30 / CD82, CD30 / CD95, CD30 / CD97, CD30 / CD122, CD30 / CD124, CD30 / CD132, CD30 / CDw137, CD20 / CD11a, CD20 / CD15, CD20 / CD18, CD20 / CD29, CD20 / CD39, CD20 / CD48, CD20 / CD55, CD20 / CD58, CD20 / CD59, CD20 / CD82, CD20 / CD95, CD20 / CD97, CD20 / CD122,CD20 / CD124, CD20 / CD132, CD20 / CDw137, CD22 / CD11a, CD22 / CD15, CD22 / CD18, CD22 / CD29, CD22 / CD39, CD22 / CD45, CD22 / CD48, CD22 / CD52, CD22 / CD55, CD22 / CD58, CD22 / CD59, CD22 / CD82, CD22 / CD95, CD22 / CD97, CD22 / CD122, CD22 / CD124, CD22 / CD132, CD22 / CDw137, MUC1 / CD11a, MUC1 / CD15, MUC1 / CD18, MUC1 / CD29, MUC1 / CD39, MUC1 / CD45, MUC1 / CD48, MUC1 / CD52, MUC1 / CD55, MUC1 / CD58, MUC1 / CD59, MUC1 / CD82, MUC1 / CD95, MUC1 / CD97, MUC1 / CD122, MUC1 / CD124, MUC1 / CD132, MUC1 / CDw137, MUC1* / CD11a, MUC1* / CD15, MUC1* / CD18, MUC1* / CD29, MUC1* / CD39, MUC1* / CD45, MUC1* / CD48, MUC1* / CD52, MUC1* / CD55, MUC1* / CD58, MUC1* / CD59, MUC1* / CD82, MUC1* / CD95, MUC1* / CD97, MUC1* / CD122, MUC1* / CD124, MUC1* / CD132, MUC1* / CDw137, PSMA / CD11a, PSMA / CD15, PSMA / CD18, PSMA / CD29, PSMA / CD39, PSMA / CD45, PSMA / CD48, PSMA / CD52, PSMA / CD55, PSMA / CD58, PSMA / CD59, PSMA / CD82, PSMA / CD95, PSMA / CD97, PSMA / CD122, PSMA / CD124, PSMA / CD132, PSMA / CDw137, CD33 / CD11a, CD33 / CD15, CD33 / CD18, CD33 / CD29, CD33 / CD39, CD33 / CD45, CD33 / CD48, CD33 / CD52, CD33 / CD55, CD33 / CD58, CD33 / CD59, CD33 / CD82, CD33 / CD95, CD33 / CD97, CD33 / CD122, CD33 / CD124, CD33 / CD132, CD33 / CDw137, MCSP / CD11a,MCSP / CD15, MCSP / CD18, MCSP / CD29, MCSP / CD39, MCSP / CD45, MCSP / CD48, MCSP / CD52, MCSP / CD55, MCSP / CD58, MCSP / CD59, MCSP / CD82, MCSP / CD95, MCSP / CD97, MCSP / CD122, MCSP / CD124, MCSP / CD132, MCSP / CDwl37, cMet / CD11a, cMet / CD15, cMet / CD18, cMet / CD29, cMet / CD39, cMet / CD45, cMet / CD48, cMet / CD52, cMet / CD55, cMet / CD58, cMet / CD59, cMet / CD82, cMet / CD95, cMet / CD97, cMet / CD122, cMet / CD124, cMet / CD132, cMet / CDwl37, EphA2 / CD11a, EphA2 / CD15, EphA2 / CD18, EphA2 / CD29, EphA2 / CD39, EphA2 / CD45, EphA2 / CD48, EphA2 / CD52, EphA2 / CD55, EphA2 / CD58, EphA2 / CD59, EphA2 / CD82, EphA2 / CD95, EphA2 / CD97, EphA2 / CD122, EphA2 / CD124, EphA2 / CD132, EphA2 / CDwl37, Endosialin / CD11a, Endosialin / CD15, Endosialin / CD18, Endosialin / CD29, Endosialin / CD39, Endosialin / CD45, Endosialin / CD48, Endosialin / CD52, Endosialin / CD55, Endosialin / CD58, Endosialin / CD59, Endosialin / CD82, Endosialin / CD95, Endosialin / CD97, Endosialin / CD122, Endosialin / CD124, Endosialin / CD132, Endosialin / CDwl37, Carboanhydrase / CD11a, Carboanhydrase / CD15, Carboanhydrase / CD18, Carboanhydrase / CD29, Carboanhydrase / CD39, Carboanhydrase / CD45, Carboanhydrase / CD48, Carboanhydrase / CD52, Carboanhydrase / CD55, Carboanhydrase / CD58, Carboanhydrase / CD59, Carboanhydrase / CD82, Carboanhydrase / CD95, Carboanhydrase / CD97, Carboanhydrase / CD122, Carboanhydrase / CD124,carbonic anhydrase / CD132, carbonic anhydrase / CDwl37, IGF-1R / CD11a, IGF-1R / CD15, IGF-1R / CD18, IGF-1R / CD29, IGF-1R / CD39, IGF-1R / CD45, IGF-1R / CD48, IGF-1R / CD52, IGF-1R / CD55, IGF-1R / CD58, IGF-1R / CD59, IGF-1R / CD82, IGF-1R / CD95, IGF-1R / CD97, IGF-1R / CD122, IGF-1R / CD124, IGF-1R / CD132, IGF-1R / CDwl37, FAP-alpha / CD11a, FAP-alpha / CD15, FAP-alpha / CD18, FAP-alpha / CD29, FAP-alpha / CD39, FAP-alpha / CD45, FAP-alpha / CD48, FAP-alpha / CD52, FAP-alpha / CD55, FAP-alpha / CD58, FAP-alpha / CD59, FAP-alpha / CD82, FAP-alpha / CD95, FAP-alpha / CD97, FAP-alpha / CD122, FAP-alpha / CD124, FAP-alpha / CD132, FAP-alpha / CDwl37, CD19 / CD11a, CD19 / CD15, CD19 / CD18, CD19 / CD29, CD19 / CD39, CD19 / CD45, CD19 / CD48, CD19 / CD52, CD19 / CD55, CD19 / CD58, CD19 / CD59, CD19 / CD82, CD19 / CD95, CD19 / CD97, CD19 / CD122, CD19 / CD124, CD19 / CD132, CD19 / CDwl37, CD52 / CD11a, CD52 / CD15, CD52 / CD18, CD52 / CD29, CD52 / CD39, CD52 / CD45, CD52 / CD48, CD52 / CD52, CD52 / CD55, CD52 / CD58, CD52 / CD59, CD52 / CD82, CD52 / CD95, CD52 / CD97, CD52 / CD122, CD52 / CD124, CD52 / CD132, CD52 / CDwl37, GD2 / CD11a, GD2 / CD15, GD2 / CD18, GD2 / CD29, GD2 / CD39, GD2 / CD45, GD2 / CD48, GD2 / CD52, GD2 / CD55, GD2 / CD58, GD2 / CD59,GD2 / CD82, GD2 / CD95, GD2 / CD97, GD2 / CD122, GD2 / CD124, GD2 / CD132, GD2 / CDwl37, CEA / CD11a, CEA / CD15, CEA / CD18, CEA / CD29, CEA / CD39, CEA / CD45, CEA / CD48, CEA / CD52, CEA / CD55, CEA / CD58, CEA / CD59, CEA / CD82, CEA / CD95, CEA / CD97, CEA / CD122, CEA / CD124, CEA / CD132, CEA / CDwl37, FR / CD11a, FR / CD15, FR / CD18, FR / CD29, FR / CD39, FR / CD45, FR / CD48, FR / CD52, FR / CD55, FR / CD58, FR / CD59, FR / CD82, FR / CD95, FR / CD97, FR / CD122, FR / CD124, FR / CD132, FR / CDwl37, Proteoglycan / CD11a, Proteoglycan / CD15, Proteoglycan / CD18, Proteoglycan / CD29, Proteoglycan / CD39, Proteoglycan / CD45, Proteoglycan / CD48, Proteoglycan / CD52, Proteoglycan / CD55, Proteoglycan / CD58, Proteoglycan / CD59, Proteoglycan / CD82, Proteoglycan / CD95, Proteoglycan / CD97, Proteoglycan / CD122, Proteoglycan / CD124, Proteoglycan / CD132, Proteoglycan / CDwl37, G250 / CD11a, G250 / CD15, G250 / CD18, G250 / CD29, G250 / CD39, G250 / CD45, G250 / CD48, G250 / CD52, G250 / CD55, G250 / CD58, G250 / CD59, G250 / CD82, G250 / CD95, G250 / CD97, G250 / CD122, G250 / CD124, G250 / CD132, G250 / CDwl37, GC182 / CD11a, GC182 / CD15, GC182 / CD18, GC182 / CD29, GC182 / CD39, GC182 / CD45, GC182 / CD48, GC182 / CD52, GC182 / CD55, GC182 / CD58, GC182 / CD59, GC182 / CD82, GC182 / CD95, GC182 / CD97, GC182 / CD122, GC182 / CD124, GC182 / CD132, GC182 / CDwl37, GT468 / CD11a, GT468 / CD15, GT468 / CD18,the group of GT468 / CD11a, GT468 / CD18, GT468 / CD29, GT468 / CD39, GT468 / CD45, GT468 / CD48, GT468 / CD52, GT468 / CD55, GT468 / CD58, GT468 / CD59, GT468 / CD82, GT468 / CD95, GT468 / CD97, GT468 / CD122, GT468 / CD124, GT468 / CD132, GT468 / CDwl37, GT512 / CD11a, GT512 / CD15, GT512 / CD18, GT512 / CD29, GT512 / CD39, GT512 / CD45, GT512 / CD48, GT512 / CD52, GT512 / CD55, GT512 / CD58, GT512 / CD59, GT512 / CD82, GT512 / CD95, GT512 / CD97, GT512 / CD122, GT512 / CD124, GT512 / CD132, and GT512 / CDwl37.

[0150] More preferably, the trifunctional bispecific antibody of the application is directed to two antigens, wherein the combination of the two antigens is selected from the group of EpCAM / CD11a, EpCAM / CD18, EpCAM / CD45, EpCAM / CD52, EpCAM / CD82, EpCAM / CD97, Her2neu / CD11a, Her2neu / CD18, Her2neu / CD45, Her2neu / CD52, Her2neu / CD82, Her2neu / CD97, MUC1 / CD11a, MUC1 / CD18, MUC1 / CD45, MUC1 / CD52, MUC1 / CD82, MUC1 / CD97, EGFR / CD11a, EGFR / CD18, EGFR / CD45, EGFR / CD52, EGFR / CD82, EGFR / CD97, EphA2 / CD11a, EphA2 / CD18, EphA2 / CD45, EphA2 / CD52, EphA2 / CD82, EphA2 / CD97, GD2 / CD11a, GD2 / CD18, GD2 / CD45, GD2 / CD52, GD2 / CD82, GD2 / CD97, CD20 / CD11a, CD20 / CD18, CD20 / CD82, and CD20 / CD97.

[0151] Preferred antibodies are heterologous trifunctional bispecific antibodies, preferably monoclonal antibodies, selected from one or more of the following combinations of isotypes:

[0152] • rat-IgG2b / mouse-IgG2a,

[0153] • rat-IgG2b / mouse-IgG2b,

[0154] • rat-IgG2b / mouse-IgG3;

[0155] • rat-IgG2b / human-IgG1,

[0156] • rat-IgG2b / human-IgG2

[0157] • rat-IgG2b / human-IgG3 [Oriental allotype G3m(st) = binds Protein A], rat-IgG2b / human-IgG4; • rat-IgG2b / rat-IgG2c;

[0158] • mouse-IgG2a / human-IgG3 [Caucasian allotype G3m(b+g) = no binding to Protein A, hereinafter

[0159] *indicates]

[0160] • mouse-IgG2a / mouse-[VH-CH1, VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3]

[0161] • mouse-IgG2a / rat-[VH-CH1, VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3]

[0162] • mouse-IgG2a / human-[VH-CH1, VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3]

[0163] • mouse-[VH-CH1, VL-CL]-human-IgG1 / rat-[VH-CH1, VL-CL]-human-IgG1-[hinge]-human

[0164] IgG3*-[CH2-CH3]

[0165] • mouse-[VH-CH1, VL-CL]-human-IgG4 / rat-[VH-CH1, VL-CL]-human-IgG4-[hinge]-human-IgG4[N-terminal region of CH2]-human-IgG3*[C-terminal region of CH2: > aa position 251]-human-IgG3*[CH3]

[0166] • rat IgG2b / mouse-[VH-CH1, VL-CL]-human-IgG1-[hinge-CH2-CH3]

[0167] • Rat-IgG2b / Mouse-[VH-CH1, VL-CL]-Human-IgG2-[hinge-CH2-CH3]

[0168] • Rat-IgG2b / Mouse-[VH-CH1, VL-CL]-Human-IgG3-[hinge-CH2-CH3, Oriental allogeneic] • Rat-IgG2b / Mouse-[VH-CH1, VL-CL]-Human-IgG4-[hinge-CH2-CH3]

[0169] • Human-IgG1 / Human-[VH-CH1, VL-CL]-Human-IgG1-[hinge]-Human-IgG3*-[CH2-CH3]

[0170] • Human-IgG1 / Rat-[VH-CH1, VL-CL]-Human-IgG1-[hinge]-Human-IgG4 [N-terminal region of CH2]-Human-IgG3* [C-terminal region of CH2: > aa position 251]-Human-IgG3* [CH3]

[0171] • Human-IgG1 / Mouse-[VH-CH1, VL-CL]-Human-IgG1-[hinge]-Human-IgG4 [N-terminal region of CH2]-Human-IgG3* [C-terminal region of CH2: > aa position 251]-Human-IgG3* [CH3]

[0172] • Human-IgG1 / Rat-[VH-CH1, VL-CL]-Human-IgG1-[hinge]-Human-IgG2 [N-terminal region of CH2]-Human-IgG3* [C-terminal region of CH2: > aa position 251]-Human-IgG3* [CH3]

[0173] Human-IgG1 / Mouse-[VH-CH1, VL-CL]-Human-IgG1-[hinge]-Human-IgG2 [N-terminal region of CH2]-Human-IgG3* [C-terminal region of CH2: > aa position 251]-Human-IgG3* [CH3]

[0174] • Human-IgG1 / Rat-[VH-CH1, VL-CL]-Human-IgG1-[hinge]-Human-IgG3*-[CH2-CH3]

[0175] • Human-IgG1 / Mouse-[VH-CH1, VL-CL]-Human-IgG1-[hinge]-Human-IgG3*-[CH2-CH3]

[0176] • Human-IgG2 / Human-[VH-CH1, VL-CL]-Human-IgG2-[hinge]-Human-IgG3*-[CH2-CH3]

[0177] • human-IgG4 / human-[VH-CH1, VL-CL]-human-IgG4-[hinge]-human-IgG3*-[CH2-CH3]

[0178] • human-IgG4 / human-[VH-CH1, VL-CL]-human-IgG4-[hinge]-human-IgG4 [N-terminal region of CH2]-human-IgG3* [C-terminal region of CH2: > aa position 251]-human-IgG3* [CH3]

[0179] • mouse-IgG2b / rat-[VH-CH1, VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3]

[0180] • mouse-IgG2b / human-[VH-CH1, VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3]

[0181] • mouse-IgG2b / mouse-[VH-CH1, VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3]

[0182] The trifunctional bispecific antibody with monovalent binding specificity used in a particularly preferred embodiment of the application has the following properties:

[0183] a) binds to a pan-leukocyte antigen selected from the group consisting of at least one member of the group of CD11a, CD15, CD18, CD29, CD39, CD45, CD48, CD52, CD55, CD58, CD59, CD82, CD95, CD97, CD122, CD124, CD132 and CDw137;

[0184] b) binds to a tumor-associated antigen on a tumor cell;

[0185] c) binds via its Fc part to an Fc receptor positive cell;

[0186] The trifunctional bispecific antibody is further preferably selected from the group of antibodies with the following isotype combinations:

[0187] • rat-IgG2b / mouse-IgG2a,

[0188] • rat-IgG2b / mouse-IgG2b,

[0189] • rat-IgG2b / human-IgG1,

[0190] Mouse-[VH-CH1; VL-CL]-human IgGl / rat-[VH-CH1, VL-CL]-human IgGl-[hinge]-human- IgG3*-[CH2-CH3]

[0191] [*= Caucasian allotype G3m(b+g) = does not bind to Protein A].

[0192] Particularly preferred are antibodies, preferably trifunctional bispecific antibodies and / or scaffold proteins against EpCAM and CD52, and the isotype combination rat- IGG2b / mouse-IgG2a. A preferred example of said trifunctional bispecific antibodies is an anti-CD52 x anti-EpCAM antibody. Preferably, said antibodies are monoclonal.

[0193] Preferably, the antibodies according to the application are monoclonal, chimeric, recombinant, synthetic, semisynthetic or chemically modified whole antibodies, which have, for example, Fv, Fab, scFv or F(ab)2 fragments.

[0194] In the methods of the application, antibodies or derivatives or fragments of human origin or antibodies modified to be suitable for use in humans (so-called "humanized antibodies") can also be used (see Shalaby et al., J. Exp. Med. 175 (1992), 217; Mocikat et al., Transplantation 57 (1994), 405).

[0195] The production of the different types of antibodies and antibody fragments described above is obvious to the person skilled in the art. The production of monoclonal antibodies, preferably of monoclonal antibodies of mammalian origin, for example of human, rat, mouse, rabbit or goat monoclonal antibodies, can be carried out using conventional methods, for example and Milstein (Nature 256 (1975), 495) as described in Harlow and Lane (Antibodies, A Laboratory Manual (1988), Cold Spring Harbor) or Galfré (Meth. Enzymol. 73 (1981), 3).

[0196] The antibodies can also be produced by recombinant DNA techniques according to techniques obvious to the person skilled in the art (see Kurucz et al., J. Immunol. 154 (1995), 4576; Hollinger et al., Proc. Natl. Acad. Sci. USA 90 (1993), 6444).

[0197] The preparation of antibodies having two different specificities, so-called bispecific antibodies, can be carried out, for example, using recombinant DNA technology, but also by means of the so-called hybridoma fusion technology (cf., for example, Milstein et al., Nature 305 (1983), 537). This technology comprises fusing hybridoma cell lines which produce antibodies having one of the desired specificities and identifying and isolating recombinant cell lines which produce antibodies having both specificities.

[0198] If trifunctional bispecific or trifunctional antibodies exhibit the properties and effects described herein, the problems underlying the present application can be overcome by using, in preferred embodiments, trifunctional bispecific or trifunctional antibodies. The present application is described in detail by way of trifunctional bispecific antibodies. However, it is to be understood that trifunctional antibodies which exhibit similar effects are also included. Although the above-mentioned term "antibody" or "scaffold protein" can refer to trifunctional bispecific antibodies, it is to be understood that it can also encompass trifunctional antibodies which exhibit similar effects.

[0199] The preparation of antibodies having three specificities, so-called trifunctional antibodies, is also suitable for solving the problems underlying the present application, for example, by coupling a third antigen-binding site having an additional specificity, for example, in the form of a "single-chain variable fragment" (scFv) to one of the IgG heavy chains of a bispecific antibody. Furthermore, recombinant techniques can be used, for example, vector-based methods for protein synthesis or oligonucleotide synthesis.

[0200] Similarly, trifunctional F(ab)2constructs can be prepared by replacing the CH2-CH3 region of the heavy chain of one of the specificities of a bispecific antibody with a scFv having a third specificity, whereas the CH2-CH3 region having the other specificity in the heavy chain can be removed, for example, by insertion of a stop codon (at the end of the "hinge" 5 region) into the encoding gene, for example, by homologous recombination.

[0201] Trifunctional single-chain antibody constructs can also be prepared in which three VH-VL regions representing three different specificities are arranged in series.

[0202] The complete bispecific antibody consists of two antibody halves (each antibody half has one H and one L immunoglobulin chain), each antibody half representing one specificity, in addition, as with normal antibodies, there is an Fc portion, which performs the well-known effector functions. They are preferably produced using the Quadrome technique. This production method is illustrated in DE-A-44 19 399. For the complete disclosure, the definition of bispecific antibodies is incorporated by reference in this document. It is understood that other production methods can be used to obtain complete bispecific antibodies, if they are in accordance with the above definition as claimed in the present invention, are feasible.

[0203] For example, using the newly developed production method, sufficient amounts of complete bispecific antibodies can be produced (Lindhofer et al., J. Immunology, 155: 219 (1995)). The combination of two bispecific antibodies against two different tumor-associated antigens on breast cancer cells (e.g. c-erb-B2, EpCAM, e.g. GA-733-2 = C215) minimizes the risk that tumor cells expressing only one of the antigens are not identified.

[0204] According to the method of the present invention, the antibodies or scaffold proteins that recognize tumor-associated antigens on tumor cells are not bound to magnetic particles or magnetic beads, or any other means of binding by magnetic particles or magnetic beads, which can have been used in the art as a tool for removing the conjugates formed by the binding of the antibodies to the tumor cells and optionally immune cells. Furthermore, the prior art describes the labeling of tumor cells or antibodies with a chromophore, such as a fluorescent dye, followed by cell sorting, such as flow cytometry. In contrast to this, the principle of the present invention is to form a three-dimensional network (conjugate) as described above, which is of a size sufficient to be retained by a filter or separated by centrifugal force, and to remove the network directly by mechanical means such as filtration or centrifugation or a combination thereof. The size of the conjugates formed by the interaction of the antibodies can be removed to some extent by filtration or centrifugation. Embodiments with magnetic beads or similar tools bound to the antibodies or tumor cells can be combined with the present invention as an indirect auxiliary means for removing the conjugates between the antibodies and the tumor cells. Furthermore, separation methods can also be combined with the present invention, such as flow cytometry methods for separating tumor cells from normal cells.

[0205] The terms "conjugate", "multicellular complex" and "aggregate" can be used interchangeably and always define a three-dimensional network between antibodies and / or scaffold proteins, tumor cells and / or immune cells and / or other tumor cells to form cross-linked tumor cells that can be removed by centrifugation or filtration or a combination thereof. The conjugate consists in particular of antibodies, tumor cells and immune cells, which are leukocytes and / or Fc receptor-positive cells.

[0206] The time used in the present application is between 10 and 240 minutes, preferably between 10 and 180 minutes, to obtain the intraoperatively recovered blood containing the cell aggregates. In the present application, the contact time is also preferably between 20 and 210 minutes, more preferably between 30 and 180 minutes, even more preferably between 30 and 120 minutes. In this respect, it was found that the time period is surprisingly critical for achieving the intended effect. When the time period is less than 10 minutes, the formation of aggregates is insufficient. When the time exceeds 180 minutes, the immune process can be initiated by the bound antibodies of the immune cells. In particular when the time exceeds 240 minutes, the onset of an unwanted immune process can be observed. The temperature is preferably room temperature, which ranges from 19 °C to 25 °C, preferably about 21 °C.

[0207] According to a preferred embodiment, the blood preparation obtained by using the method of the present application is then re-administered into the patient from which the initial blood sample was obtained, without causing severe side effects.

[0208] Contacting antibodies and / or scaffold proteins with IBS

[0209] Preferably, if any antibody is not able to bind to an immune cell, but only to the same or a different tumor-associated antigen, it is essential that there is a cross-linking between at least two different tumor cells in order to obtain an association between the antibody and the at least two tumor cells.

[0210] The terms "binding", "multicellular complex" and "aggregate" can be used interchangeably and always define a three-dimensional network between antibodies, scaffold proteins, tumor cells and / or immune cells and / or other tumor cells to form cross-linked tumor cells which can be removed by centrifugation or filtration or a combination thereof. The binding specifically consists of antibodies, tumor cells and leukocytes.

[0211] Preferably, the at least one antibody is contacted with the intraoperatively obtained blood recovery for a time period which is sufficient to cross-link tumor cells and optionally immune cells and / or other tumor cells to obtain a binding. Preferably, the tumor cells and the immune cells are present in the intraoperatively obtained blood recovery. The tumor cells can be specifically recognized by the at least one antibody or the at least one scaffold protein.

[0212] The time required to achieve the cross-linking can be determined by the person skilled in the art using routine methods. The time used in the present application is between 10 and 240 minutes, preferably between 10 and 180 minutes. The contact time is preferably between 10 and 118 minutes, more preferably between 20 and 90 minutes, even more preferably between 30 and 60 minutes. The temperature is preferably room temperature, which can range from 19 °C to 25 °C, preferably about 21 °C.

[0213] The amount of antibody that has to be administered in order to obtain the described conjugate comprising the antibody can be determined by the person skilled in the art by routine measures. The amount of antibody and / or scaffold protein to be administered usually depends on the binding affinity of the antibody and / or scaffold protein to the tumor antigen. Preferably, the further amount is 1-20 pg / LIBS, more preferably 1-10 pg / LIBS, 1-5 pg / LIBS or 1-2 pg / LIBS.

[0214] Preferably, at least two different antibodies and / or scaffold proteins are applied, which differ from each other in their tumor antigen specificity or pan leukocyte surface marker specificity, provided that at least one antibody is within the limits of claim 1.

[0215] Removal of tumor cells and antibody / scaffold protein conjugate from IBS

[0216] Finally, the conjugate is removed from the blood recovery (IBS) obtained during the procedure in order to obtain an IBS which is essentially free of tumor cells. The removal of the cell aggregates is preferably achieved by centrifugation, filtration or a combination thereof. In a preferred method, at least one centrifugation step is included and more preferably at least one filtration step.

[0217] The filtration is preferably a leukocyte reduction or depletion filtration, wherein all leukocytes contaminated with the tumor cells are removed entirely or essentially when the red blood cells pass through the filter and are collected. The filter can be chosen from a mesh filter with a pore size between 20 pm and 40 pm, which retains the conjugate formed by the method of the present application, as well as e.g. fibrin strands and dead cell clumps. Red blood cells with a size of about 8 pm can pass through the filter. Microaggregates filters are preferably used for the reinfusion of autologous blood collected during or after the procedure. For receiving leukocyte-reduced red blood cells, it is also possible to use standard blood management filters with a pore size between 170 pm and 260 pm for filtration.

[0218] A review describing cell preservation devices including separation methods is Carless PA, Henry DA, Moxey AJ, O’Connell D, Brown T, Fergusson DA, Cell salvage for minimizing perioperative allogeneic blood transfusion, 2010, The Cochrane Collaboration, John Wiley & Sons, Ltd., which is fully incorporated by reference.

[0219] The Pall SQ 40S transfusion filter is an example of a microaggregate blood filter which is only used to receive the part of the blood containing red blood cells.

[0220] Preferably, centrifugation is performed by a density gradient centrifugation step, followed by washing with e.g. physiological saline for a period of time and centrifugation at a speed sufficient to remove the tumor cell containing conjugate and to separate red blood cells from the tumor cell conjugate and white blood cells.

[0221] Preferably, filtration is performed for a period of time sufficient to at least substantially remove the tumor cell containing conjugate and to separate red blood cells from the tumor cell conjugate and white blood cells.

[0222] In a preferred embodiment, the red blood cell concentrate free or substantially free of contaminating tumor cells is used without the addition of physiological saline.

[0223] A further filtration step can be used to remove residual conjugate and / or white blood cells.

[0224] Since the basic white blood cell filtration technology differs from traditional micron-sized filtration procedures, including clogging problems, this advanced tumor cell removal strategy appears feasible.

[0225] One of the advantages of the present invention is that it significantly reduces the time, complexity, selectivity, operational strength involved in removing tumor cells compared to removing tumor cells by destroying them with antibodies or by removing them with magnetic beads or radiation or cell sorting methods, making it feasible to remove tumor cells within the limited time span available during a tumor surgery.

[0226] Other advantages of the present invention include a significant reduction of tumor cells in the red blood cell concentrate produced from the surgical blood and a significant reduction of pro-inflammatory cytokines produced in the surgical field by the surgical trauma. Furthermore, residual trifunctional antibodies can be reduced to an insignificant amount in the final EC concentrate for reinfusion.

[0227] Preferably, the blood product obtained by using the method of the present invention is subsequently re-administered to the patient from which the initial blood sample was obtained. BRIEF DESCRIPTION OF DRAWINGS

[0228] Figure 1 : shows the principle of formation of large multicellular complexes induced by trifunctional antibodies used in the present invention, e.g. anti-CD52 x anti-EpCAM antibodies. The complex formation allows removal of tumor cells by specific processing steps (e.g. centrifugation and filtration) of the intraoperative blood sample device.

[0229] Figure 2: Schematic representation of representative antibody formats with the ability to form multicellular complexes consisting of tumor cells and peripheral blood leukocytes (i.e. immune cells). Figure taken from Jin P and Z Zhu. In: Bispecific Antibodies. Kontermann RE (ed.), Springer Heidelberg Dordrecht London New York, pp. 151-170 (2011) and slightly modified. Only antibodies falling under claim 1 of the present invention belong to the present invention; all other antibody formats are only for reference.

[0230] Figure 3 : Exemplary embodiment of the method of the present invention using a trifunctional bispecific antibody (BsAb) of the present invention, e.g. an anti-CD52 x anti-EpCAM antibody.

[0231] Figure 4 : Viable cell density (VCD) and viability (VIA) of fed-batch cultures. F1-F4 - 1 L flask replicates.

[0232] Figure 5 : AC chromatogram of PGB0005 bispecific antibody purification.

[0233] Figure 6 : PGB0005 bispecific antibody treated with DTT-light chain.

[0234] Figure 7 : PGB0005 bispecific antibody treated with DTT-heavy chain.

[0235] Figure 8 : PGB0005 bispecific antibody treated with PNGase F and DTT-heavy chain.

[0236] Figure 9 : SDS-PAGE analysis of purified PGB0005 bispecific antibody. Lanes 1 and 12 - FB-blank, lanes 2-6 - reducing conditions, lanes 7-12 - non-reducing conditions, lanes 4 and 9 - PNGase F enzyme, lanes 2 and 11 - non- deglycosylated PGB0005 bispecific antibody, lanes 3, 5, 6, 7, 8 and 10 - deglycosylated PGB0005 bispecific antibody.

[0237] Figure 10 : Fc gamma receptor binding activity of PGB0005 bispecific antibody.

[0238] Figure 11A and Figure 11B : EpCAM binding activity of PGB0005 bispecific antibody: (a) includes all data points, and (b) includes only data points with a concentration of 10 nM. Figure 11A ) includes all data points, and (b) includes only data points with a concentration of 10 nM.Figure 11B Remove single hook effect data points.

[0239] Figure 12A and Figure 12B PGB0005 bispecific antibody and ( Figure 12A ) buffer solution with pH = 6.1 and ( Figure 12B CD52 binding activity in a solution buffer at pH 7.4. BsAb-FITC anti-mouse and BsAb-FITC anti-rat antibodies were used in the assay. Detailed Implementation

[0240] An exemplary embodiment for carrying out the invention is described below. Those skilled in the art who employ this method will inevitably acquire the claimed benefits. Various modifications can be made based on this example without departing from the invention.

[0241] During IBS, multicellular complexes are removed by centrifugation and / or filtration, and anti-EpCAM-mediated tumor cell removal is performed.

[0242] The expression profile of the transmembrane glycoprotein EpCAM, classified by tissue microarray staining, appears in cancers across various tumor types (Table 3). This widespread EpCAM expression pattern represents an interesting marker of cancer, which may have significant implications not only for therapeutic applications but also for improving IBS protocols during cancer surgery. However, it is noteworthy that most soft tissue tumors and all lymphomas are EpCAM-negative.

[0243] Table 3. Tissue microarrays: EpCAM expression profiles of various tumor entities.

[0244]

[0245]

[0246] 1 Data is from Went et al., Brit. J. Cancer 94:128, 2006.

[0247] 2 Data from Spizzo et al., Gyneological Oncology 103:483, 2006.

[0248] #Score 1-4; EpCAM expression was defined by calculating the product of the total staining score (0-4) and the intensity score (0-3). The proportion score describes the estimated proportion of positively stained tumor cells (0, none; 1, <10%; 2, 10-50%; 3, 50-80%; 4, >80%). The intensity score represents the estimated staining intensity (0, no staining; 1, weak; 2, moderate; 3, strong).

[0249] Score >4; the total expression score is between 0 and 12, therefore EpCAM overexpression is defined as total score >4.

[0250] Example 1

[0251] 1. Obtaining anti-CD52 x anti-EpCAM tri-functional bispecific antibodies

[0252] In the backbone of the trifunctional bispecific antibody, the anti-lymphocyte paratope is replaced by the paratope of the anti-CD52 specific variable region binding to leukocytes. Crowe et al. published the sequence of the anti-CD52 specific variable region (H+L IgG chains) (Clin Exp Immunol, vol. 87 1992, Fig. 2).

[0253] The antibody encoding genes representing the heavy and light Ig genes of the anti-EpCAM half antibody and the heavy and light Ig genes representing the anti-CD52 half antibody were transferred into a CHO cell line. The secreted specific anti-EpCAM and anti-CD52 trifunctional antibodies from the supernatant of the transfected CHO cells in culture were purified by standard chromatography.

[0254] To verify the functionality of the two binding arms, flow cytometry analysis was performed on two different cell lines expressing either EpCAM or CD52.

[0255] 2. Evaluation of the binding of the tri-functional bispecific anti-CD52 x anti- EpCAM antibodies to different target antigens

[0256] Antibody binding was assessed by FACS analysis. 5 x 105 5 Target cells (CD52 positive Jurkat cells or EpCAM positive HCT-8 cells) were incubated with the indicated antibody concentrations for 60 min at 2-8°C. The target cells were resuspended in PBS buffer. Then, the cells were washed twice and the bound antibody on the cells was detected using a fluorescently labeled (FITC) rat anti-mouse IgG (Jurkat cells) or mouse anti-rat IgG (HCT-8 cells) secondary detection antibody (Dianova). The positively stained cells were evaluated using a FACS Calibur cytometer (Becton Dickinson).

[0257] 3. Evaluation of the binding of the tri-functional bispecific anti-CD52 x anti- EpCAM antibodies to Fc gamma receptors

[0258] Antibody binding was assessed by FACS analysis. 5 x 105target cells (Fc gamma receptor positive THP-1 cells) were incubated with the indicated antibody concentrations at 2-8°C for 60 min. The target cells were resuspended in PBS buffer. Then, the cells were washed twice and the antibody binding on the cells was detected with a fluorescently labeled (FITC) F(ab)2goat anti-mouse IgG F(ab)2secondary detection antibody (Dianova). The positively stained cells were assessed using a FACS Calibur cytometer (Becton Dickinson). 5 Antibody binding was assessed by FACS analysis. 5 x 105target cells (Fc gamma receptor positive THP-1 cells) were incubated with the indicated antibody concentrations at 2-8°C for 60 min. The target cells were resuspended in PBS buffer. Then, the cells were washed twice and the antibody binding on the cells was detected with a fluorescently labeled (FITC) F(ab)2goat anti-mouse IgG F(ab)2secondary detection antibody (Dianova). The positively stained cells were assessed using a FACS Calibur cytometer (Becton Dickinson).

[0259] Example 2

[0260] 1. Target molecule structure

[0261] PGB0005 bispecific antibody is a rat / mouse triomab designed to target CD52 and EpCAM receptors and their variable regions. The original variable region of the rat half antibody has been replaced by a region targeting CD52, which is derived from the Campath sequence described in the literature. The Catumaxomab mouse half antibody targeting the variable region of EpCAM was not modified. The gene sequences of both half antibodies were optimized for CHO expression.

[0262] 2. Batch cultivation

[0263] Method:

[0264] PGB0005 stable cell pool 5 was cultivated in Excell Advanced fed-batch medium in four 1 L flasks (working volume 300 ml) with an initial cell density of 0.5 x 10 6 From day 3 of the cultivation process, cell enhancement 7a and 7b feed media were added at 3% and 0.3% of the working volume, respectively, per day.

[0265] Results:

[0266] At day 9 of the cultivation process, the cell density reached about 23 x 10 6 cells / ml. The cell viability decreased below 90% at day 11 and the culture was harvested at day 12 with an average viable cell density of 23 x 10 6 cells / ml and an average viability of 86% ( Figure 4 ).

[0267] 3. Purification

[0268] Method:

[0269] One liter of culture supernatant was purified by isocratic affinity chromatography. MabSelect Sure resin was used with a column volume of 42 ml. 0.1 M sodium citrate was used as the elution buffer and the conditions for separation of the antibody components were as follows: pH 5.8 for elution of the parental rat antibody, pH 5.15 for elution of the target PGB0005 bispecific antibody, and pH 3.5 for elution of the parental mouse antibody.

[0270] Results:

[0271] The target bispecific antibody was successfully separated from the two parental antibodies Figure 5 ).

[0272] 4. Quality analysis

[0273] Prior to starting the fed-batch production of the described samples, the identity of the protein produced by PGB0005 stable cell pool 5 was confirmed by LC-MS. Purified PGB0005 bispecific antibody samples from fed-batch production were analyzed by SDS-PAGE and tested for Fcy receptor binding activity, EpCAM binding activity, and CD52 binding activity.

[0274] 4.1. LC-MS analysis of samples produced by purified stable cell pools 5

[0275] Method:

[0276] PGB0005 bispecific antibody samples produced in flask culture and purified by AC chromatography were analyzed by LC-MS. Prior to LC-MS analysis, the samples were subjected to two different treatments: 1) PNGase F treatment and DTT treatment for deglycosylation after denaturation, and 2) DTT treatment after denaturation. The samples were then injected into a BEH C4 column for separation and analyzed by a Q-Tof mass spectrometer.

[0277] Theoretical molecular weight information for the target proteins is shown in Table 1. Theoretical molecular weight information after post-translational modifications is shown in Tables 2 and 3.

[0278] Name MW (Da) anti-CD52 light chain 23175.9 anti-EpCAM light chain 24424.8* anti-CD52 heavy chain 49850.4 anti-EpCAM heavy chain 50021.8

[0279] Table 4. Expected (unreduced) molecular weight values for PGB0005 bispecific antibody components.

[0280] Name MW (Da) anti-CD52 heavy chain 49850.4 N-terminal pQ -17 - lysine C-terminal -128.2 + G0F 1445.3 Expected value 51150.5

[0281] Table 5. Expected molecular weight of anti-CD52 heavy chain with post-translational modifications. N-terminal pQ - N-terminal pyroglutamine modification, -Lys C-terminal - C-terminal lysine loss, +G0F - G0F glycan.

[0282] Name MW (Da) anti-EpCAM heavy chain 50021.8 N-terminal polyethylene -18 - lysine C-terminal -128.2 + G0F 1445.3 + G1 F 1607.5 Expected value + G0F 51320.9 Expected value + G1 F 51483.1

[0283] Table 6 Expected molecular weights of anti-EpCAM heavy chains with post-translational modifications. N-terminal pE - N-terminal pyroglutamic acid modification, -Lys C-terminal - C-terminal lysine loss, +G0F - G0F glycan, +G1F - G1F glycan.

[0284] Name MW (Da) anti-CD52 heavy chain 49850.4 N-terminal pQ -17 - lysine C-terminal -128.2 Expected MW 49705.2

[0285] Table 7 Expected molecular weights of anti-CD52 heavy chains with post-translational modifications but without N-glycans. N-terminal pQ - N-terminal pyroglutamine modification, -Lys C-terminal - C-terminal lysine loss.

[0286] Results:

[0287] In the sample without PNGase F treatment, the anti-CD52 light chain and the anti-EpCAM light chain were identified by MS ( Figure 6 ). The experimental molecular weight of the anti-CD52 light chain was 23177.0 Da, which well corresponded to the theoretical molecular weight of 23175.9 Da (Table 4). The experimental molecular weight of the anti-CD52 light chain was 24426.0 Da, which well corresponded to the theoretical molecular weight of 24424.8 Da (Table 1).

[0288] The anti-CD52 glycosylated heavy chain was identified with a molecular weight of 51156.0 Da ( Figure 7 ), which well corresponded to the theoretical value of 51150.5 Da for the anti-CD52 heavy chain when considering post-translational modifications (Table 5). Two MS peaks of 51156.0 Da and 51487.0 Da were identified ( Figure 7 ), which corresponded to the expected molecular weights of the anti-EpCAM heavy chain with G0F glycosylation (51320.9 Da) and G1F glycosylation (51483.1 Da), respectively (Table 6). The difference between the expected value and the observed value for the GoF glycosylated anti-EpCAM heavy chain was about 6 Da, and the difference between the expected value and the observed value for the G1F glycosylated anti-EpCAM heavy chain was about 4 Da, which might be caused by the opening of disulfide bonds due to DTT treatment. Since the mass difference between the anti-EpCAM heavy chain with G0F glycosylation and the anti-CD52 chain with G1F glycosylation was less than 10 Da, their signals might interfere with each other.

[0289] In Figure 8 , the observed peak of 49712.0 Da was consistent with the expected molecular weight of 49705.2 Da for the anti-CD52 heavy chain (Table 7). This difference of about 6 Da might be caused by the opening of disulfide bonds due to DTT treatment.

[0290] 4.2. SDS-PAGE analysis

[0291] Method:

[0292] The purified PGB0005 sample was divided into two batches: 1) non-deglycosylated and 2) deglycosylated (treated with PNGase F). Analysis was performed by SDS polyacrylamide gel electrophoresis under reducing and non-reducing conditions, respectively.

[0293] Results:

[0294] Two heavy chain bands with a molecular weight of approximately 50 kD were visible in lane 2 (non-deglycosylated sample) and lanes 3, 5 and 6 (deglycosylated sample). The molecular weight of the heavy chain band in lane 2 (non-deglycosylated) was lower than the heavy chain bands in lanes 3, 5 and 6 (deglycosylated). Figure 9

[0295] In lanes 2, 3, 5 and 6, two light chain bands with a molecular weight of approximately 25 kD were visible.

[0296] In lanes 7, 8, 10 and 11, two major bands with a molecular weight of approximately 150 kD and 200 kD, respectively, were visible. The molecular weight of the bands in lane 11 (non-deglycosylated) was lower than in lanes 7, 8 and 10 (deglycosylated).

[0297] In lanes 7, 8, 10 and 11, three additional bands with a molecular weight of approximately 120 kD, 85 kD and 22.5 kD, respectively, were visible.

[0298] 4.3. Fc gamma receptor binding activity

[0299] Method:

[0300] The Fcγ receptor binding activity of the purified bispecific antibody sample of PGB0005 was tested by flow cytometry (FACS).

[0301] The density of the THP-1 cell culture suspension was 4 x 10 6 The working sample concentration range for the PGB0005 bispecific antibody was 0.0017-100 μg / ml in 50 μl per well.

[0302] The binding reaction was performed at 2-8°C for 1 h. The dilution buffer was pH 7.4 PBS and the washing buffer was pH 7.4 PBS containing 2% fetal calf serum.

[0303] FITC-goat anti-mouse F(ab')2 specific fragment was used as the detection antibody.

[0304] The antibody detection reaction was performed at 2-8°C for 30 minutes.​

[0305] Results:

[0306] PGB0005 bispecific antibody showed Fcγ receptor binding activity ( Figure 10 ).

[0307] 4.4. EpCAM binding activity

[0308] Method:

[0309] The EpCAM binding activity of the bispecific antibody sample purified from PGB0005 was detected by flow cytometry (FACS).

[0310] HCT-8 adherent cells were cultured at a density of 1.5 × 10⁻⁶. 6 Cells / ml, 100 μl per well. The working sample concentration range for the PGB0005 bispecific antibody is 0.0017-100 μg / ml, 50 μl per well.

[0311] The conjugation reaction was carried out at room temperature for 1 hour. PBS with 2% fetal bovine serum at pH 7.4 was used as a dilution buffer and also as a cell washing solution.

[0312] FITC-mouse anti-mouse antibody (H+L) was used as the detection antibody.

[0313] The antibody detection reaction was carried out at 2-8℃ for 30 minutes.

[0314] Results:

[0315] PGB0005 bispecific antibody showed EpCAM binding activity ( Figure 11A and Figure 11B Due to the hook effect, a single data point with a concentration of 100 μg / ml was removed.

[0316] 4.5. CD52 binding activity

[0317] Method:

[0318] The purified PGB0005 bispecific antibody sample was dissolved in PBS buffer at two different pH values ​​(7.4 and 6.1), and CD52 binding was detected by flow cytometry (FACS). Two different detection antibodies were used. Two replicate experiments were performed.

[0319] The density of the Ramos cell culture suspension is 4 × 10⁻⁶. 6 Cells / ml, 50 μl per well. The working sample concentrations used for the PGB0005 bispecific antibody are shown in the table below:

[0320] Repetition Buffer pH BsAb working concentration 1 PBS pH 7.4 0.0203 - 1196.9 pg / mL 1 PBS pH 6.1 0.0172 - 1014.0 pg / mL 2 PBS pH 7.4 0.0172 - 1017.2 pg / mL 2 PBS pH 6.1 0.0147 - 867.6 pg / mL

[0321] Table 8. Concentration of working samples for CD52 binding assay experiments.

[0322] The binding reaction was performed at 2-8°C for 1 hour. The sample buffer (PBS, pH 7.4 or pH 6.1) was also used as dilution buffer, and PBS, pH 7.4 plus 2% fetal bovine serum as washing buffer.

[0323] FITC-mouse anti-rat (H+L) and FITC-rat anti-mouse (H+L) were used as detection antibodies.

[0324] The antibody detection reaction was performed at 2-8°C for 30 minutes.

[0325] Results:

[0326] PGB0005 bispecific antibodies showed CD52 binding activity at pH 6.1 and 7.4 Figure 12A and Figure 12B Compared to mouse anti-rat detection antibodies, rat anti-mouse detection antibodies appeared to bind stronger to the target bispecific antibodies. The results of two independent experiments were consistent.

[0327] Summary

[0328] Using the cell saver system and the trispecific bispecific anti-CD52 x anti- EpCAM antibody purified cancer patient blood samples did not show or essentially not detectable residual tumor cells (0) in the red blood cell concentrates. Furthermore, the cytokines secreted during surgery were significantly reduced in the red blood cell concentrates after the procedure and there were no safety issues for the patients. Importantly, even using a very sensitive ELISA method (detection limit: 250 pg / ml) no or essentially no residual anti-CD52 x anti-EpCAM antibody was detected in the produced red blood cell concentrates, thereby confirming the safety of potential patients receiving these purified autologous red blood cell concentrates.

[0329] Conclusion

[0330] Due to the potential risk of undetected and undetected viral infections as well as other risks associated with allogeneic blood transfusions, the growing demand and economic demands, intraoperative blood recovery is increasingly gaining the attention of hospital blood management programs. However, the use of intraoperative autotransfusion in major cancer surgery is limited due to the possibility of tumor cells reinfusing into the patient and thus leading to an increased rate of recurrence.

[0331] It has been found that the anti-CD52 x anti-EpCAM antibody mediates the formation of multicellular complexes with its binding capacity to cancer cells, leukocytes and accessory cells, which can be simply removed by centrifugation and / or leukocyte filtration (a routine step of leukocyte depletion). The present application shows the potential anti-CD52 x anti-EpCAM antibody-mediated tumor cell / leukocyte removal capacity during intraoperative blood salvage by using automated devices such as the cell or saver® or the like. The in vitro application of the anti-CD52 x anti-EpCAM antibody represents the use as a medical device and as a medical product, respectively, during intraoperative blood salvage procedures in cancer surgery.

[0332] Starting from the data supported by these experiments, it is evident to the person skilled in the art that this experimentally proven concept can also be effectively transferred to other monoclonal antibodies, provided that they are able to interact with tumor cells and, optionally, with immune cells and / or other tumor cells, to form aggregates or conjugates that can be removed by, for example, filtration or centrifugation. For these reasons, the present application is not limited to trifunctional bispecific antibodies recognizing the tumor-associated antigen EpCAM and the pan-leukocyte marker CD52, such as the anti-CD52 x anti-EpCAM antibody, and the specific isotype combinations described herein; after the concept of the present application has been described herein, it can be easily transferred to other antibodies and tumor-associated antigens described herein, as long as they are able to recognize tumor-associated antigens and to form removable conjugates.

[0333] In summary, the application of the method of the present application, for example by using the anti-CD52 x anti-EpCAM antibody, to minimize the risk of residual autologous tumor cells will be a prerequisite for performing IBS during cancer surgery.

[0334] The present invention provides a faster and more efficient method of removing tumor cells from intraoperative recovered blood compared to conventional methods. The antibodies, particularly trifunctional bispecific antibodies, disclosed herein bind to leukocytes, tumor cells and Fc gamma receptor positive accessory cells, forming a three-dimensional network of antibodies, tumor cells and immune cells and further optionally or additionally other tumor cells. The trifunctional bispecific antibodies are capable of binding to specific pan leukocyte antigens, which allows virtually up to 100% of the lymphocytes of various types of blood cells, including T cells, to bind to tumor cells. Thus, compared to other known methods, e.g. methods in which antibodies mediate the binding of only T cells to tumor cells, the trifunctional bispecific antibodies disclosed herein mediate a significantly increased number of blood cells, including leukocytes and lymphocytes, to interact with tumor cells in intraoperative recovered blood, thereby allowing a more efficient removal of tumor cells. In this respect, the present invention is particularly advantageous in cases where the T cell content in the peripheral blood of a cancer patient is low, because other known methods which rely on the binding of T cells to tumor cells can not be efficient in removing tumor cells, whereas the present invention is not limited in this respect, because the trifunctional bispecific antibodies also bind to various other types of blood cells in addition to T cells.

[0335] During the process of incubating the antibodies in blood for removing tumor cells, cytokines can be released from the immune cells and in some cases accumulate in the red blood cell concentrate, which can cause an immune reaction upon transfusion of such red blood cell concentrate to a patient, despite the washing step. In other known methods for removing tumor cells from intraoperative recovered blood, in which T cells are mediated to bind to tumor cells, particularly when this binding is mediated via the T cell specific antigens CD3 or CD28, a large amount of cytokines can be released into the blood within a short time, leading to a severe inflammatory reaction in the patient. In this respect, although the use of the trifunctional bispecific antibodies of the present invention can also theoretically lead to the release of cytokines, the extent and speed of this release should be significantly reduced, thereby significantly reducing the risk of a severe inflammatory reaction caused by cytokines released in the surgical blood.

[0336] Furthermore, as described above, the antibodies, particularly the trifunctional bispecific antibodies, disclosed herein mediate a large number of blood cells, including leukocytes and lymphocytes, to interact with tumor cells. Thus, compared to other known methods, in which e.g. only T cells are mediated to interact with tumor cells, the antibodies disclosed herein have the potential to form larger conjugates and / or aggregates with blood cells. Furthermore, the antibodies disclosed herein bind to leukocyte antigens which are not expressed on the surface of red blood cells, thus avoiding binding to red blood cells. In particular, the antibodies of the present invention which bind to CD52 have been shown not to bind to red blood cells. Thus, the method of the present invention efficiently separates tumor cells from red blood cells prior to reintroducing the purified blood into the patient.

[0337] Hospital bedside / laboratory blood transfusion filter (www.pall.com / medical6595.asp)

[0338]

[0339]

Claims

1. An in vitro method for removing tumor cells from blood recovered during surgery, comprising the following steps: - Provide intraoperatively salvaged blood that may contain tumor cells outside the human body; - Contact the blood recovered during the operation with at least one trifunctional bispecific antibody. The at least one trifunctional bispecific antibody described herein has the following characteristics: a) The conjugate is selected from at least one of the group consisting of CD29, CD39, CD48, CD52, CD55, CD58, CD59, CD82, CD95, CD97, CD122, CD124, CD132 and CDw137; b) Binds to tumor-associated antigens on tumor cells; c) It binds to Fc receptor-positive cells via its Fc moiety. The at least one trifunctional bispecific antibody is capable of forming a three-dimensional network with at least tumor cells and leukocytes in the blood. In this process, the at least one trifunctional bispecific antibody is contacted with the intraoperatively recovered blood for 10-240 minutes, which is sufficient to crosslink tumor cells containing the tumor-associated antigen and leukocytes containing the panleukocyte antigen, as well as other tumor cells, to obtain conjugates and / or aggregates containing the at least one trifunctional bispecific antibody, wherein the tumor cells, the other tumor cells, and the leukocytes may be present in the intraoperatively recovered blood, and wherein the tumor cells are specifically recognized by the at least one trifunctional bispecific antibody; and - Mechanically remove the conjugates and / or aggregates from the blood recovered during the procedure.

2. The in vitro method according to claim 1, characterized in that, The trifunctional bispecific antibody is in contact with the blood recovered during the operation for 10-180 minutes.

3. The in vitro method according to claim 1, characterized in that, The trifunctional bispecific antibody is selected from a group of antibodies having the following isotype combinations: Rat-IgG2b / Mouse-IgG2a Rat-IgG2b / Mouse-IgG2b Rat-IgG2b / human-IgG1 Mouse - [VH-CH1; VL-CL] - Human - IgG1 / Rat - [VH-CH1, VL-CL] - Human - IgG1 - [hinge] - Human - IgG3* - [CH2-CH3] *=Caucasian allotype G3m(b+g)=Does not bind to protein A.

4. The in vitro method according to claim 1, characterized in that, At least two different antibodies are used, whose tumor antigen specificity and / or panleukocyte antigen specificity differ from each other.

5. The in vitro method according to claim 1, characterized in that, The conjugate consists of antibodies, tumor cells, and leukocytes.

6. The in vitro method according to claim 1, characterized in that, The dosage of the at least one trifunctional bispecific antibody is 1 μg to 20 μg per liter of blood recovered during surgery.

7. The in vitro method according to claim 6, characterized in that, The dosage of the at least one trifunctional bispecific antibody is 1 to 10 μg per liter of blood recovered during surgery.

8. The in vitro method according to claim 7, characterized in that, The dosage of the at least one trifunctional bispecific antibody is 1 to 5 μg per liter of blood recovered during surgery.

9. The in vitro method according to claim 8, characterized in that, The dosage of the at least one trifunctional bispecific antibody is 1 to 2 μg per liter of blood recovered during surgery.

10. The in vitro method according to claim 1, characterized in that, It further includes at least one of the following steps: a) Before adding the at least one trifunctional bispecific antibody, mix the blood recovered during the operation with at least one anticoagulant; b) Separating red blood cells from the conjugates and / or aggregates, as well as blood components, by centrifugation; and c) Filter the mixture to remove any remaining conjugates and / or aggregates, as well as any residual cell complexes.

11. The in vitro method according to claim 10, characterized in that, Step b) involves separating red blood cells from the conjugates and / or aggregates, as well as blood components, by density gradient centrifugation.

12. The in vitro method according to claim 1, characterized in that, The incubation time between the at least one trifunctional bispecific antibody and the blood recovered during the operation is between 10 and 90 minutes.

13. The in vitro method according to claim 12, characterized in that, The incubation time between the at least one trifunctional bispecific antibody and the blood recovered during the operation is between 20 and 60 minutes.

14. The in vitro method according to claim 12, characterized in that, The incubation is carried out at a temperature between 19 and 25°C.

15. The in vitro method according to claim 14, characterized in that, The incubation is carried out at room temperature.

16. The in vitro method according to claim 1, characterized in that, The conjugates and / or aggregates are removed by centrifugation, filtration, or a combination thereof.

17. The in vitro method according to claim 1, characterized in that, In a separate step, filters that adsorb or remove leukocytes are used to remove leukocytes and / or cell complexes containing tumor cells.

18. The in vitro method according to claim 1, characterized in that, The tumor cells are derived from epithelial, blood, or neuroectodermal tumors.

19. The in vitro method according to claim 1, characterized in that, In addition, a trifunctional bispecific antibody is administered that binds to tumor-associated antigens on T cells and tumor cells and binds to Fc receptor-positive cells via its Fc portion.

20. The in vitro method according to any one of claims 1 to 19, characterized in that, The at least one trifunctional bispecific antibody has the property of binding to two different tumor-associated antigens.

21. Uses of trifunctional bispecific antibodies with the following characteristics: a) Binds to panleukocyte antigen, selected from at least one of the group consisting of CD29, CD39, CD48, CD52, CD55, CD58, CD59, CD82, CD95, CD97, CD122, CD124, CD132 and CDw137; b) Binds to tumor-associated antigens on tumor cells; c) It binds to Fc receptor-positive cells via its Fc moiety. The antibody is capable of forming a three-dimensional network with tumor cells and leukocytes contained in the blood in an in vitro method according to any one of claims 1 to 19.

22. The use according to claim 21, characterized in that, The at least one trifunctional bispecific antibody has the property of binding to two different tumor-associated antigens.

23. A trifunctional bispecific antibody for treating tumors or cancer, characterized in that, Includes the following steps: - Provide intraoperatively salvaged blood that may contain tumor cells outside the human body; - Contact the blood recovered during the procedure with the antibody, wherein the antibody contains the following properties: a) Conjugates panleukocyte antigen, selected from at least one of the group consisting of CD29, CD39, CD48, CD52, CD55, CD58, CD59, CD82, CD95, CD97, CD122, CD124, CD132 and CDw137; b) Binds to tumor-associated antigens on tumor cells; c) It binds to Fc receptor-positive cells via its Fc moiety. The antibody can form a three-dimensional network with at least tumor cells and white blood cells in the blood. The antibody is in contact with the intraoperative blood for 10-240 minutes, which is sufficient to crosslink tumor cells containing the tumor-associated antigen and leukocytes containing the panleukocyte antigen, as well as other tumor cells, to obtain conjugates and / or aggregates containing the antibody, wherein the tumor cells, the other tumor cells and the leukocytes may be present in the intraoperative blood, and the tumor cells are specifically recognized by the antibody. and - Mechanically remove the conjugates and / or aggregates from the blood recovered during the procedure.

24. The trifunctional bispecific antibody according to claim 23, characterized in that, The trifunctional bispecific antibody is in contact with the blood recovered during the operation for 10-180 minutes.

25. The trifunctional bispecific antibody according to claim 23, characterized in that, The trifunctional bispecific antibody has the property of binding to two different tumor-associated antigens.

Images