Method of removing tumor cells from allogeneic erythrocyte concentrates

Abstract

The present invention relates to an ex vivo method of preparing purified erythrocyte concentrates (EC) or a whole blood product for transfusion, comprising a step of contacting an allogeneic blood collection with a bispecific and / or a multispecific antibody or a combination of parental antibodies in a blood salvage device (BSD). Said EC or whole blood product can be derived from a mixture of blood of two or more donors, and the purified EC or the whole blood product can be introduced to a subject who is a recipient in need of said purified EC or the whole blood product.

Description

[0001] Method of removing tumor cells from allogeneic erythrocyte concentrates

[0002] Technical field

[0003] The present invention relates to a method for preparing purified erythrocyte concentrates (EC) or a whole blood product.

[0004] Background

[0005] Erythrocyte concentrates (EC), also known as red cell concentrate are by far the most common blood component used in transfusion medicine. They are obtained from whole blood and consist mainly of red blood cells (erythrocytes). Erythrocyte concentrates can be obtained directly from the donor by means of erythrocyte-apheresis or by fractionation of whole blood in a blood donation center. In the latter case, centrifugation is used to separate the whole blood into erythrocytes, "buffy coat" and cell-free blood plasma. After separation of the supernatant (buffy coat and plasma), the erythrocytes remain, which are then suspended in a stabilizer solution. Leukocyte depletion is achieved by additional filtration. ECs are frequently used both in autologous and allogeneic (homologous) blood transfusion.

[0006] A circulating tumor cell (CTC) is a type of cell which has shed into lymphatics or vasculature from a primary tumor, whereby said CTC cells can be carried around in the blood circulation around the whole body. The CTC cells are characterized in that they can seed tumors, i.e. contributing to the growth of additional tumors in distant organs. Thus, the removal of said CTC before transfusion is critical for ensuring the safety of the subject receiving transfusion. Particularly, after obtaining erythrocyte concentrates (EC), the CTC still contained therein needs to be sufficiently removed.

[0007] In addition to the tumor cells in the blood circulation, under certain circumstances EC or a whole blood product produced from collections of blood donors can contain other unwanted nucleated cells, preferably lymphocytes or leukocytes of various origins. Said whole blood product is a well-known and established term in transfusion medicine, which includes, for example, erythrocytes, plasma, and thrombocytes and leukocytes. To remove such unwanted cells from already produced ECs can be challenging. The reason is that currently available filter systems cannot remove such cells sufficiently to avoid possible complications as e.g. immunosuppression (by allogeneic lymphocytes) or transfer of cancer from donor to recipient. When recipient is immunocompromised due to various reasons, allogeneic tumor cells can under certain circumstances establish tumor growth in the recipient (Molodysky&Grant, March 2021 , Asian Pac. J Cancer Prev., 22: 641 -649). Current guidelines for blood donation allow cancer patients to donate blood after a recurrence-free time period of 5 years. However, circulating tumor cells in peripheral blood can still be present in recurrence-free patients, which could lead to tumor cell contamination of ECs. Another source of tumor cell containing ECs are donors which have developed cancer but still have no cancer diagnosis. For these reasons, it would be useful to develop and establish a technology to remove unwanted nucleated cells, preferably lymphocytes and / or leukocytes as well as tumor cells, from already produced ECs, before infusion to recipients especially immuno-compromised or-suppressed recipients.

[0008] Problems to be solved by the present Invention

[0009] The aim of the present invention is to provide a method of preparing purified EC or a whole blood product for transfusion. The presently disclosed method involves using a bispecific and / or a multispecific antibody or a combination of parental antibodies, which are able to bind unwanted nucleated cells, preferably tumor cells or cancer cells, more preferably lymphocytes, leukocytes or EpCAM positive tumor cells or cancer cells of hematological origin with sufficient affinity to hold such cells back in a blood salvage device (BSD), preferably a cell saver device or machine, to generate a purified EC or a whole blood product before reaching the recipients body during transfusion.

[0010] All of preferred embodiments in the present application involving said purified EC also apply to a whole blood product, and a person skilled in the art with a mind willing to understand and in light of common general knowledge would know which preferred embodiments involving said purified EC would apply to a whole blood product.

[0011] The presently disclosed method involves using a bispecific and / or a multispecific antibody or a combination of parental antibodies in combination with a BSD, preferably a cell saver machine.

[0012] The presently disclosed method refers to an ex vivo method for preparing purified erythrocyte concentrates (EC) for transfusion, comprising a step of contacting an allogeneic blood collection with a bispecific and / or a multispecific antibody or a combination of parental antibodies in a BSD.

[0013] Through binding with above-stated antibodies, the presently disclosed method can remove unwanted nucleated cells, preferably tumor cells or cancer cells, more preferably lymphocytes, leukocytes or EpCAM positive tumor cells and / or cancer cells of hematological origin and / or cancer cells of neuroectodermal origin that display sufficient affinity with above-stated antibodies, by aggregating said different cell types including cancer and / or tumor cells in a BSD, e.g. a cell saver machine, as commonly used in the art. The presently disclosed method can be applied to produce a purified EC from a blood donor. The purified EC can be introduced into another subject who is the recipient in need of said EC, and said recipient can even be immuno-compromised or-suppressed recipients. In this respect, with the presently disclosed method, unwanted nucleated cells, preferably tumor cells, cancer cells and lymphocytes or leukocytes of various origins can be significantly reduced from the EC, whereby e.g. tumor metastases would be reduced or avoided, and recurrence of tumor or immunomodulation would be suppressed.

[0014] Description of the Drawings

[0015] Figure 1 : Schematic illustration of a possible crosslink between CD52 positive leukocytes, EpCAM positive carcinoma cells and CD64 (Fc-gamma receptor type I) positive immune cells by a trifunctional antibody (e.g.: anti-CD52 x anti-EpCAM) leading to 3-dimensional cell complexes / aggregates.

[0016] Detailed Description of the Invention and Preferred Embodiments

[0017] The following discussion is included for purposes of describing the present invention and illustrating preferred embodiments thereof.

[0018] Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure 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 disclosure, 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 explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0019] The present invention concerns:

[0020] (1) An ex vivo method for preparing purified erythrocyte concentrates (EC) for transfusion, comprising a step of contacting an allogeneic blood collection with a bispecific and / or a multispecific antibody or a combination of parental antibodies in a blood salvage device (BSD).

[0021] (2) The ex vivo method of (1 ), wherein said BSD is a cell saver machine. (3) The ex vivo method of (1 ) or (2), wherein said allogeneic blood collection is a mixture of blood collections from two or more different donors.

[0022] (4) The ex vivo method of any one of (1 ) to (3), wherein the contacting step is performed by incubating said allogeneic blood collection with said bispecific and / or said multispecific antibody or said combination of parental antibodies for predetermined time period, so that said bispecific and / or the multispecific antibody or the combination of parental antibodies bind with unwanted nucleated cells, preferably lymphocytes, leukocytes and / or tumor cells.

[0023] (5) The ex vivo method of any one of (1 ) to (4), wherein erythrocyte concentrates (EC) are generated in the BSD by a centrifugation step and / or a filtration step, or wherein erythrocyte concentrates (EC) are generated in the BSD by a filtration step without any centrifugation step.

[0024] (6) The ex vivo method of (5), comprising a further step of washing said bispecific and / or the multispecific antibody or the combination of parental antibodies that bind with unwanted nucleated cells, preferably lymphocytes, leukocytes and / or tumor cells, wherein said washing step is performed before the centrifugation step and / or the filtration step, or after the centrifugation step and / or the filtration step.

[0025] (7) The ex vivo method of any one of (1 ) to (6), wherein unwanted nucleated cells, preferably lymphocytes, leukocytes and / or tumor cells, are removed or their amount is reduced from said EC, more preferably at least 95% of tumor cells being removed and / or at least 80% of leukocytes, lymphocytes being removed.

[0026] (8) The ex vivo method of any one of (1 ) to (7), wherein the bispecific and / or the multispecific antibody is a trifunctional antibody, wherein the three arms of said trifunctional antibody respectively comprises the following properties: a) binding to a leukocyte or a tumor-associated antigen on a tumor cell or leukocyte; b) binding to a tumor-associated antigen on a tumor cell; c) binding via its Fc-portion to an Fc-receptor positive cell, wherein preferably the tumor cell or the leukocyte in a) are of hematological origin such as leukemias or lymphomas, and b) of epithelial or neuroektodermal origin like such as carcinoma or melanoma or sarkoma, small cell lung cancer or glioma.

[0027] (9) The ex vivo method of (8), wherein the three arms of said trifunctional antibody respectively comprises the following properties: a) binding to a tumor-associated antigen on a tumor cell; b) binding to a tumor-associated antigen on a tumor cell which is different from the antigen in a); c) binding via its Fc-portion to an Fc-receptor positive cell.

[0028] (10) The ex vivo method of (8) or (9), wherein the arm of the trifunctional antibody corresponding to the property a) can bind to a pan-leukocyte antigen, which is selected from a group consisting of CD11 a, CD15, CD18, CD29, CD39, CD45, CD48, CD52, CD55, CD58, CD59, CD82, CD95, CD97, CD122, CD124, CD132, and CDw137, preferably the pan-leukocyte antigen is CD52.

[0029] (11 ) The ex vivo method of (8) or (9), wherein the arm of the trifunctional antibody corresponding to the property a) can bind to T cell through a T cell surface antigen, and wherein the T cell surface antigen is selected from a group consisting of CD2, CD3, CD4, CD5, CD6, CD8, CD28, CD40L and CD44, CD80, CD86, preferably the T cell surface antigen is CD3.

[0030] (12) The ex vivo method of any one of (8)-(11 ), wherein the arm of the trifunctional antibody corresponding to the property b) can bind to a tumor associated antigen which is selected from the group consisting of: EpCAM, Her2neu, EGFR, CD30, CD38, CD79, CD123, CD133, PD-L1 CD20, CD22, MUC-1 , MUC-16, MUC-17, MUC1 * with changed glycosylation pattern, PSMA, CD33, MCSP, cMet, EphA2, Endosialin, Carboanhydrase IX, IGF-1 R, FAP-alpha, CD19, BCMA, GD2, CEA, proteoglycans, G250, GC182, GT468, GT512, GD3, GM1 , GM2, GM3, fucosyl-GM1 , globo-H, S1 P, Cer, and Gg3, preferably the tumor associated antigen is EpCAM or CD20 or GD2.

[0031] (13) The ex vivo method of (8) or (9), wherein the arms of the trifunctional antibody corresponding to the property a) and b) can bind to a tumor associated antigen which is selected from the group consisting of: EpCAM, Her2neu, EGFR, CD30, CD38, CD123, CD133, PD-L1 CD20, CD22, MUC1 , MUC1 * with changed glycosylation pattern, PSMA, CD33, MCSP, cMet, EphA2, Endosialin, Carboanhydrase IX, IGF-1 R, FAP- alpha, CD19, BCMA, GD2, CEA, FR, proteoglycans, G250, GC182, GT468, GT512, GD3, GM1 , GM2, GM3, fucosyl-GM1 , globo-H, S1 P, Cer, and Gg3, preferably the tumor associated antigen is EpCAM or CD20 or GD2.

[0032] (14) The ex vivo method of any one of (8)-(13), wherein the trifunctional antibody comprises a binding site in its Fc-portion for Fey receptor type I, II and / or III.

[0033] (15) The ex vivo method of any one of (8)-(14), wherein the trifunctional antibody can bind to monocytes, macrophages, dendritic cells, natural killer cells and / or activated neutrophils via their Fey receptor type I, II and / or III. Other preferred embodiments are described in the following description. Further preferred features of the invention can be taken from the claims.

[0034] Parental antibodies are well-known for a skilled person in the art, as they are specifically related to bispecific or multispecific antibody. Said parental antibodies may refer to antibodies which are used to generate said bispecific or multispecific antibody. Said parental antibodies can be naturally occurring or genetically constructed antibodies.

[0035] The BSD, preferably the cell saver device or machine are commonly used for intraoperative blood salvage, also known as cell salvage, and a person skilled in the art knows how to access to said BSD or cell saver device or machine without any undue burden.

[0036] BSD is a device that can be used in autologous blood transfusion to recover blood lost during surgery wherein it can collect the blood lost from the patient, clean and / or separate certain blood fractions like e.g. erythrocytes, platelets and / or thrombocytes and reintroduce the blood or separated blood fractions into the same patient.

[0037] The inventors of the present invention have now found out that a BSD can be used for preparing purified erythrocyte concentrates (EC) for transfusion, wherein an allogeneic blood collection is contacted with a bispecific and / or a multispecific antibody or a combination of parental antibodies in a blood salvage device (BSD).

[0038] In the present invention, a blood salvage device normally used for autologous blood transfusion during surgery can be used for contacting allogeneic blood collection with said bispecific and / or said multispecific antibody or said combination of parental antibodies.

[0039] In this regard, a “blood salvage device” is well known in the art, and it can also mean a device of “Mechanical Autotransfusion”, a device of “Intraoperative Blood Salvage”, or a device of “Autologous Blood Transfusion”, which are all well known to a person skilled in the art.

[0040] Preferably, the blood collection is a mixture of blood collections from two or more different donors which belong to the same blood group and Rhesus factor and / or fulfill criteria to be called as “matched” in the field of transfusion medicine. In blood transfusion, a "match" refers to the compatibility between the donor's and the recipient's blood types. This compatibility is crucial for the safety and effectiveness of the transfusion, as it prevents adverse reactions in the recipient's body. Blood typing is based on the presence or absence of certain antigens on the surface of red blood cells, with the most significant being the ABO and Rh (Rhesus) blood group systems.

[0041] In ABO system, individuals can have blood type A, B, AB, or O, depending on the presence of A or B antigens on their red blood cells. Type AB individuals can receive blood from any ABO type (universal recipient), while type O individuals can donate to any ABO type (universal donor) because their red blood cells do not have A or B antigens that could cause an immune response.

[0042] In Rh System, blood is classified as either Rh-positive (+) or Rh-negative (-) based on the presence or absence of the RhD antigen. Rh-negative individuals can only receive Rh-negative blood to avoid an immune reaction, while Rh-positive individuals can receive blood that is either Rh-positive or Rh-negative.

[0043] A successful "match" for a blood transfusion requires compatibility in both the ABO and Rh systems to avoid immune responses such as hemolytic reactions, where the recipient's immune system attacks the transfused blood cells, potentially leading to serious complications. Beyond ABO and Rh, there are other blood group systems and factors considered in certain cases to ensure the best match and minimize the risk of adverse reactions.

[0044] Preferably, above two or more different donors do not include the patient himself.

[0045] Preferably, the volume of said allogeneic blood collection for contacting with above-stated antibodies is at least 300 ml, preferably 300-1200 ml, 500-1200 ml, 700-1200 ml, or 900-1200 ml, or preferably 800-3000 ml, 1000-3000 ml, 1 100-3000 ml, 1100-2800 ml, 1 100-2600 ml, or 1100-2400 ml, more preferably 800-2200 ml.

[0046] Preferably, the predetermined time period for incubating said allogeneic blood collection with said bispecific and / or said multispecific antibody or said combination of parental antibodies is at least 5 minutes, more preferably said predetermined time period being 5-10 minutes. Still more preferably, said predetermined time period is 5-15 minutes, 5-20 minutes, 5-30 minutes, 5-40 minutes, 5-80 minutes, 5-90 minutes, 5-120 minutes, 15-90 minutes, 20-80 minutes, or 30-40 minutes.

[0047] A predetermined time period of 5 minutes would result in a sufficient binding of said bispecific and / or the multispecific antibody or the combination of parental antibodies bind with unwanted nucleated cells, preferably lymphocytes, leukocytes and / or tumor cells. A prolonged time period of more than 5 minutes would improve the binding of said bispecific and / or the multispecific antibody or the combination of parental antibodies bind with unwanted nucleated cells, preferably lymphocytes, leukocytes and / or tumor cells.

[0048] Preferably, the above predetermined time period for incubating said allogeneic blood collection with said bispecific and / or said multispecific antibody or said combination of parental antibodies should not be longer than 150 minutes, which otherwise could lead to an unwanted stimulation of immune cells, resulting in e.g. unspecific and / or uncontrolled cytokine release, whereby a disease such as inflammation would be caused.

[0049] Preferably, erythrocyte concentrates (EC) are generated in the BSD by a centrifugation step and a filtration step, wherein the centrifugation step is preceding the filtration step, wherein more preferably, a further step of washing said bispecific and / or the multispecific antibody or the combination of parental antibodies that bind with unwanted nucleated cells, is performed before the centrifugation step or after the filtration step.

[0050] Preferably, erythrocyte concentrates (EC) are generated in the BSD by a centrifugation step and a filtration step, wherein the filtration step is preceding the centrifugation step, wherein more preferably, a further step of washing said bispecific and / or the multispecific antibody or the combination of parental antibodies that bind with unwanted nucleated cells, is performed before the filtration step or after the centrifugation step.

[0051] Preferably, erythrocyte concentrates (EC) are generated in the BSD by a filtration step, without any centrifugation step. In this regard, said BSD can be a filtration-based autotransfusion device, preferably as described by Mansour et al. (Anesthesiology 2021 ; 135:246-57), e.g. i-SEP device. Preferably, said BSD is a cell saver machine or a filtration system, as disclosed by Mansour et al. (Anesthesiology 2021 ; 135:246- 57). More preferably, the use of said BSD is also the same as that described by Mansour et al. (Anesthesiology 2021 ; 135:246-57). In this regard, compared to the device involving centrifugation, it is found that the bispecific and / or the multispecific antibody or the combination of parental antibodies can be efficiently applied to the filtration-based autotransfusion device, and the sufficiently removal of unwanted nucleated cells can be achieved, wherein both blood cells (i.e. erythrocytes) and / or platelets are recovered with minimal hemolysis without compromising cell integrity.

[0052] Preferably, when erythrocyte concentrates (EC) are generated in the BSD by a filtration step without any centrifugation step, a further step of washing said bispecific and / or the multispecific antibody or the combination of parental antibodies that bind with unwanted nucleated cells, is performed before the filtration step, more preferably as described by Mansour et al. (Anesthesiology 2021 ; 135:246-57). The involvement of said washing step would further improve the efficiency of removal of unwanted nucleated cells as well as the removal of unbound antibody. Preferably, the amount of above-stated antibody for contacting with said allogeneic blood collection is 0.25- 10gg, 1.0-10.0 gg, 2.0-9.0 gg, 2.0-8.0 gg, 2.0-7.0 gg, or 2.0-6.0 gg, more preferably, 1.50-4.50 gg, 2.50- 9.50 gg, 2.50-8.50 gg, 2.50-7.50 gg, 2.50-6.50 gg, or 2.50-5.50 gg.

[0053] Preferably, said EC is isolated from above-stated antibodies binding with unwanted nucleated cells such as lymphocytes, leukocytes and / or tumor cells, preferably through gravity by using a leukocyte depletion filter, e.g. a Haemonetics RS1 or Fresenius BioR flex leukocyte depletion filter or a Puriblood LDF.

[0054] Preferably, when washing step is performed as disclosed above, the presently disclosed method also sufficiently removes one or more of activated and non-activated coagulation factors, complement factors, free hemoglobin, platelets, leukocytes (up to 99% reduction depending on the system), heparin, antibiotics, fat (up to 99.8% reduction depending on the system), and inflammatory mediators.

[0055] Preferably, the nucleated cells are reduced by at least 80% in the purified EC, compared to EC without being subjected to the method of present invention. Said nucleated cells can be unwanted nucleated cells for transfusion that can contain tumor cells, lymphocytes and / or leukocytes. Said nucleated cells can be part of a blood product from a donor, which is e.g. for transfusion into another subject as a recipient.

[0056] The time period for contacting said bispecific and / or multispecific antibody or a combination of parental antibodies with said allogeneic blood collection can be 10 min to 2.5 h, 30 min to 2.5 h, 45 min to 2 h, or 30min h to 1 ,5 h, preferably 20 min to 1 h, more preferably, 15min to 1 h.

[0057] Preferably, above unbound antibody is washed away by buffer-solutions during a washing step well-known in the art.

[0058] BSD devices used can vary from simple, inexpensive, sterile bottles filled with anticoagulant to expensive, sophisticated, high speed cell washing devices (e.g. Medtronic Sequestra 1000, Cobe BRAT 2, Medtronic Autolog, Haemonetics Cell Saver-5® and Fresenius CATS®; Bentzien el al., Anaesthesist 49: 505, 2000; Serrick 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).

[0059] Cell saver machine can be from Livanova or Sorin - XTRA, CellSaver Elite (i.e. Haemonetics), autoLog IQ (i.e. Medtronic), or C.A.T.S.plus(i.e. Fresenius). A review describing the blood salvage device (BSD) 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.

[0060] As described in above review from Carless PA et al., in respect of cell salvage devices, various types of cell salvage (autotransfusion) systems were studied, wherein exemplified systems can be listed as follows. ABTrans autologous re-transfusion system

[0061] Atrium 2050

[0062] Atrium 2550 in-line autotransfusion drainage system

[0063] Autovac postoperative orthopaedic autotransfusion canister

[0064] Bard cardiotomy reservoir

[0065] Bellovac ABT autotransfusion system

[0066] Beijing PerMed Biomedical Engineering Company

[0067] Bentley Catr hard shell cardiotomy reservoir

[0068] BIODREN autotransfusion system

[0069] BRAT-2 Cell Saver

[0070] CATR 3500 cardiotomy reservoir

[0071] Cell Trans system (Summit Medical)

[0072] ConstaVac CBC system

[0073] ConstaVac CBCII system

[0074] COBE Bayler rapid autotransfusion system

[0075] Dideco Compact

[0076] Dideco Electra system

[0077] Dideco 742 cardiotomy reservoir

[0078] Dideco 797 reinfusion system (Sorin Biomedical)

[0079] DONOR system (Van Straten Medical)

[0080] Electromedic Autotrans AT-100

[0081] Electromedics BT-795

[0082] Flow-Gard 6200 (Baxter)

[0083] Fresenius continuous autotransfusion system (C.A.T.S)

[0084] Gish Orthofuser Biomedical autotransfusion system

[0085] Haemonetics Cell Saver

[0086] Haemonetics Cell Saver 3

[0087] Haemonetics Cell Saver 3 Plus

[0088] Haemonetics Cell Saver 4 Haemonetics Cell Saver 5

[0089] Haemonetics Haemolite cell washer

[0090] Haemonetics Haemolite-2

[0091] Medtronic Autolog system

[0092] Ortho-Evac system

[0093] Pleur-evac autotransfusion system

[0094] Shiley hardshell cardiotomy reservoir

[0095] Solcotrans Cell Saver

[0096] Solcotrans Orthopedic Plus system

[0097] Solcotrans Orthopedic system

[0098] Sorenson ATS (autotransfusion system)

[0099] Terumo TE-171 system (Terumo)

[0100] Preferably, above exemplified systems of cell salvage devices can be used as cell saver machine in the presently disclosed method.

[0101] Preferably, BSD and cell saver machine are interchangeable in the present application.

[0102] Through contacting said allogeneic blood collections with said bispecific and / or multispecific antibody or a combination of parental antibodies, unwanted nucleated cells such as lymphocytes and / or leukocytes as well as tumor cells can be bound by antibodies leading to cell complexes and so held back or separated in the device by centrifugation and / or filtering systems like e.g. leukocyte depletion filters or column containing beads, whereas erythrocytes can pass the device and subsequently can reach the recipients blood circulation by transfusion of erythrocyte concentrates.

[0103] Also through contacting said allogeneic blood collections with said bispecific and / or multispecific antibody or a combination of multispecific / bispecific and / or a combination of parental antibodies, unwanted nucleated cells such as lymphocytes and / or leukocytes as well as tumor cells can be bound and aggregated by the applied antibodies and so hold back or separated in the blood salvage device (BSD), preferably a cell saver machine, containing centrifugation steps and / or optionally a filtering system preferably a leukocyte depletion filter (i.e. LDF), whereas erythrocytes can pass the device will finally be collected as a filtered erythrocyte concentrate and reach the recipients blood circulation by transfusion.

[0104] Preferably, the bispecific and / or the multispecific antibody is trifunctional antibody, wherein the three arms of said trifunctional antibody respectively comprises the following properties: a) binding to a leukocyte or a tumor-associated antigen on a tumor cell; b) binding to a tumor-associated antigen on a tumor cell; c) binding via its Fc-portion to an Fc-receptor positive cell, wherein more preferably the tumor cell in a) is of hematological origin such as leukemias or lymphomas, and b) of epithelial or neuroektodermal origin like such as melanoma or sarkoma, small cell lung cancer or glioma.

[0105] Preferably, above-disclosed trifunctional antibody is selected from the group consisting of a bispecific, trispecific, tetraspecific and multispecific antibody, more preferably a trifunctional bispecific antibody.

[0106] In the present invention, the term “tumor cell” refers to any cell which can divide relentlessly and form solid tumors or flood the blood with abnormal cells. Preferably, in the present invention, the tumor cells present in the EC can be from epithelial, haematological or neuroectodermal tumors.

[0107] Examples of tumors included in the present invention (but not limited thereto) comprise 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 carcinoma, ductal carcinoma and lobular breast carcinoma), lung cancers, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytomas sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor, seminoma, bladder carcinoma, and CNS tumors (such as a glioma, astrocytoma, medulloblastoma, craniopharyrgioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma and retinoblastoma). Further examples include epithelial tumors, hematological tumors such as leukemias and lymphomas and neuroectodermal tumors.

[0108] Preferably, in the ex vivo method of the present invention, said tumor cells are from epithelial, hematological or neuroektodermal tumors.

[0109] In the present invention, the “Fc-receptor cell” refers to cells with Fc-receptor present on the cell surface. Preferably, the “Fc-receptor positive cell” refer to one or more of monocyte, macrophage, dendritic cell, natural killer cell, neutrophil and eosinophilic cell.

[0110] Preferably, the Fc-receptor positive cell is Fc-gamma receptor positive cells.

[0111] In the present invention, “erythrocyte concentrate” refers to packed red cells. Preferably, said trifunctional antibody is Catumaxomab, a trifunctional antibody with binding specificities anti-EpCAM x anti-CD3, as well as a Fc-portion able to bind Fc-gamma receptor positive cells like e.g. monocytes or NK (natural killer) cells.

[0112] Preferably, said trifunctional antibody is resulted from a combination of parental anti-EpCAM and anti-CD3 antibodies, whereas the anti-CD3 antibodies can be replaced by other T cell binding antibodies like e.g. anti- CD4, anti-CD8, anti-CD2, anti-CD6 antibodies.

[0113] Preferably, said trifunctional antibody is a bispecific antibody with specificities anti-EpCAM x anti-CD52. Such a bispecific antibody binds to EpCAM positive tumor cells as well as all lymphocytes / leukocytes and cancer cells of hematological origin, as CD52 is a pan-leukocyte marker which is also expressed on the majority of cancer cells of hematological origin like e.g leukemias, lymphomas and / or other hematological cancers.

[0114] Preferably, said trifunctional antibody is resulted from a combination of parental anti-EpCAM antibodies and anti-CD52 antibodies, whereas the anti-CD52 antibodies can be replaced by other pan-leukocyte antibodies such as CD1 1 a, CD15, CD18, CD29, CD39, CD45, CD48, CD55, CD58, CD59, CD82, CD95, CD97, CD122, CD124, CD132, or CDw137.

[0115] Preferably, said trifunctional antibody is resulted from a combination of other tumor and / or hematological cancer binding antibodies as e.g. anti-CD20, anti-CD19, anti-BCMA, anti-CD30, anti-CD33, anti-CD38, anti- CD123, anti-CD133 and anti-PD-L1 together with anti-CD2, anti-CD3, anti-CD4, anti-CD6 and anti-CD8 antibodies and / or anti-pan leukocyte specific antibodies like e.g. anti-CD52.

[0116] Preferably, said trifunctional antibody is a combination of bispecific antibodies which bind to e.g. anti-CD20 x anti-CD3 and / or anti-EpCAM x anti-CD3 and / or anti-EpCAM x anti-CD52 and / or anti-CD19 x anti-CD3 and / or anti-CD33 x anti-CD3 and / or anti-CD38 x anti-CD3 and / or anti-CD33 x anti-CD20 and / or anti-CD33 x anti-CD38.

[0117] Preferably, said trifunctional antibody is a bispecific antibody with specificities anti-EpCAM x anti-CD52. Such a bispecific antibody binds to EpCAM positive tumor cells as well as all lymphocytes / leukocytes and cancer cells of hematological origin, as CD52 is a pan-leukocyte marker which is also expressed on the majority of cancer cells of hematological origin like e.g leukemias, lymphomas and / or other hematological cancers. Preferably, said trifunctional antibody is a bispecific antibody with specificities anti-EpCAM x anti-CD52, in combination with a bispecific or monospecific antibody binding to cancer cells of neuroektodermal origin like e.g. melanoma, sarcoma, small cell lung cancer and glioma with e.g. specificities anti- GD2 x anti-CD3 or anti-GD2.

[0118] The antibodies used in the present invention can be trifunctional antibodies. Preferably, the trifunctional antibody in the present invention can be bi-, tri-, tetra- and multispecific antibodies. The trifunctional antibody disclosed below refers to trifunctional bispecific antibody. However, if the tri-, tetra- and multispecific antibodies also exhibit the same properties or effects, said trifunctional antibody can also refer to a trifunctional tri-, tetra- and multispecific antibody as used herein.

[0119] Generally, a bispecific antibody is defined as an antibody capable of binding to two different types of antigens preferably via its variable region; a trispecific antibody is characterized by binding to three different types of antigens preferably via its variable region; a tetraspecific antibody is characterized by binding to four different types of antigens preferably via its variable region while a multispecific antibody is defined as being capable of binding multiple different types of antigens preferably via its variable region. As one specific example, the trifunctional bispecific antibody anti-CD3 x anti-EpCAM is defined by binding to the tumor- associated antigen EpCAM on the one hand and to the T cell surface antigen CD3 on the other hand as well as with accessory cells by its Fc part.

[0120] Generally, the bi-, tri-, tetra- and multispecific antibodies described above may be monovalent, divalent, trivalent, tetravalent or multivalent. An antibody with a monovalent binding property is defined as an antibody which is capable of binding to one tumor-associated antigen. A bivalent monoclonal antibody is defined as an antibody which is capable of binding to two tumor-associated antigens or one tumor-associated antigen and one immune cell-associated antigen. A trivalent monoclonal antibody is defined as an antibody which is capable of binding to 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. A tetravalent monoclonal antibody is defined as an antibody which is capable of binding to four different tumor-associated antigens or two different tumor-associated antigens - each having two identical antigen binding arms - or two / three tumor-associated antigens and one immune cell-associated antigen or two tumor-associated antigens and two immune cell-associated antigens. A multivalent monoclonal antibody is defined as an antibody which is capable of binding to one or more tumor-associated antigens and / or one or more immune cell associated antigen. The term "binding to a tumor-associated antigen" is defined as binding to an epitope of said tumor-associated antigen on a tumor cell. Only those antibodies having the trifunctional bispecific format as described by claim 1 are covered by the invention. All other antibodies are described only for information purposes. General description of bifunctional or trifunctional antibodies are described by Kontermann RE (ed.), Springer Heidelberg Dordrecht London New York, pp. 1 -28 (201 1 ) having bispecific or trispecific (trifunctional formats with bivalent, trivalent and tetravalent) binding properties to one tumor-associated antigen and to one or more surface antigens of leukocytes (i.e. cells of the immune system) are of importance for this patent application.

[0121] • Bispecific antibody formats with bivalent antigen binding features: e.g. scFv (e.g. BiTE class), Db, scDb, dsDb, DART, dAba / VHHa, knob-into-holes derivates, SEED-IgG, heteroFc-scfv, Fab-scFv, CrossMabs

[0122] • Bi- (tri-) specific antibody formats with trivalent antigen binding features: e.g. triple body, DNL-F(ab)s, SCFV2-CHI / CL, dAbs, Fab-scFv2, IgG-scFab

[0123] • Bi- (tri-) specific antibody formats with tetravalent antigen binding features: e.g. IgG-scFv, scFv-IgG, scFv-Fc, F(ab’)2-scFv2, sDb-Fc, scDb-Ch3, Db-Fc, scFv2-H / L, DVD-lg, tandAb, scFv-dhlx-scFv, dAb2-lgG, two-in-one mAb, mAb2, dAb-IgG, dAb-Fc-dAb.

[0124] Additional antibodies to be used according to the invention are described in the following references, which are hereby incorporated by reference:

[0125] Muller D and RE Kontermann. In: Bispecific Antibodies. Kontermann RE (ed.), Springer Heidelberg Dordrecht London New York, pp. 83-100 (2011 ) scFv (BiTE)

[0126] Baeuerle PA, Zugmaier G and D Ruttinger. In: Bispecific Antibodies. Kontermann RE (ed.), Springer Heidelberg Dordrecht London New York, pp. 273-288 (201 1 )

[0127] DVD-lg

[0128] 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 )

[0129] DNL-derivatives 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 )

[0130] Two-in-one antibodies

[0131] Koeing P and G Fuh. In: Bispecific Antibodies. Kontermann RE (ed.), Springer Heidelberg Dordrecht London New York, pp. 187-198 (2011 )

[0132] CrossMabs

[0133] Schaefer et al. Proc. Natl. Acad. Sci. USA 108: 1 1187 (2011 )

[0134] Preferably, two or more trifunctional antibodies with different specificities can be combined for mediating the associates of tumor cells and immune cells, e.g. leukocytes.

[0135] The trifunctional antibody according to the present invention may bind to a T cell via a T cell surface antigen selected from a group consisting of CD2, CD3, CD4, CD5, CD6, CD8, CD28, CD40L and CD44, CD80, CD86. It means that the antibody for use according to the present invention preferably comprises a paratope which can recognize and bind to an epitope of a T cell surface antigen selected from the group consisting of CD2, CD3, CD4, CD5, CD6, CD8, CD28, CD40L and CD44, CD80, CD86. This specificity preferably promotes the recruitment of T cells.

[0136] Preferably, the T cell surface antigen is CD3. It means that the antibody for use according to the present invention further preferably comprises a paratope which can recognize and bind to an epitope of CD3.

[0137] Catumaxomab, an example for a trifunctional antibody, binds EpCAM-positive tumor cells and CD3-positive T-cells through its 2 specific binding sites. Catumaxomab also recruits FcyR type I, Ila and Ill-positive accessory cells via binding of its intact fragment crystallizable (Fc) region resulting in a trifunctional mode of action. The primary mode of action of Catumaxomab in the context of the present invention consists of the physical aggregation of tumor cells and lymphocytes / accessory cells and the subsequent removal of the cell aggregates by centrifugation and filtration

[0138] In this context, especially for the present invention, the investigation of Arva and Andersson is of relevance showing that proinflammatory cytokines IL1 (3, TNF-a, IL-6, IL-8, IFN-y and IL-12 were not secreted before 4 hours after stimulation. This time period is necessary after stimulation for de novo synthesis of cytokines which is the regular pathway (Arva E, Andersson B. Kinetics of cytokine release and expression of lymphocyte cell-surface activation markers after in vitro stimulation of human peripheral blood mononuclear cells with Streptococcus pneumoniae. Scand J Immunol. 1999:49(3):237-243.).

[0139] In a preferred embodiment, the antibodies used in the present invention are monoclonal antibodies. This is specifically true for the trifunctional bispecific antibodies disclosed herein in detail.

[0140] Proteins having relatively defined three-dimensional structures are commonly referred to as protein scaffolds. These protein scaffolds may be used as reagents for the design of artificially engineered antibodies. These scaffolds typically contain one or more regions which are amenable to specific or random sequence variation, and such sequence randomization is often carried out to produce libraries of proteins from which the desired antibody scaffolds may be selected. Such scaffolds are particularly useful in the field of antibody design.

[0141] These antibody scaffolds are non-immunoglobulin proteins which mimic properties of a monoclonal antibody with respect to its binding activity to for instance tumor cells and immune cells. Scaffolds often include loops or domains which form the binding side of said antibody scaffold. These antibody mimics may be utilized for the purpose of designing proteins which are capable of binding to virtually any compound of interest. This directed evolution approach results in the production of antibody-like molecules with high affinities for antigens of interest. In addition, those scaffolds may be used to display defined exposed loops (e.g. loops previously randomized and selected on the basis of antigen binding) in order to direct evolution of molecules that bind to such introduced loops. Methods on how to obtain antibody-like scaffold proteins are known in the art. The following describes one possible approach for obtaining an antibody-like scaffold protein.

[0142] A first screening method, useful for the isolation or identification of randomized or mutated proteins of interest, involves: (a) contacting a compound of interest with a candidate protein, the candidate protein being a derivative non-antibody protein including a domain having an immunoglobulin-like fold, the nonantibody protein deriving from a reference protein by having a mutated amino acid sequence wherein the non-antibody protein binds with a Kd at least as tight as 1 microM to a compound that is not bound as tightly by the reference protein, wherein the contacting is carried out under conditions that allow compound-protein complex formation; and (b) obtaining, from the complex, the derivative protein that binds to the compound.

[0143] The second screening method is for isolating or identifying a compound which binds to a tumor-associated protein of interest. This method begins with a non-antibody protein including a domain having an immunoglobulin-like fold and deriving from a reference protein by having a mutated amino acid sequence, wherein the non-antibody protein binds with a Kd at least as tight as 1 pM to a compound that is not bound as tightly by the reference protein. This derivative protein is then contacted with a candidate compound (tumor-associated antigen or an epitope thereof), wherein the contacting is carried out under conditions that allow compound-protein complex formation, and the compound which binds to the derivative protein is obtained from the complex. Again, this general technique may be carried out with any protein.

[0144] Further methods of obtaining non-antibody proteins which bind to compounds of interest (tumor-associated antigen or an epitope thereof) are described as follows. One such method involves: (a) providing a nonantibody scaffold protein including an immunoglobulin-like fold, wherein the scaffold protein does not bind to the compound with a Kd as tight as 1 micro M; (b) generating mutated derivatives of the non-antibody scaffold protein, thereby producing a library of mutated proteins; (c) contacting the library with the compound; (d) selecting from the library at least one derivative protein which binds to the compound with a Kd at least as tight as 1 pM; and (e) optionally repeating steps (b)- (d) substituting for the non-antibody scaffold protein in repeated step (b) the product from the previous step (d). Again, this general technique may be carried out with any protein.

[0145] The so produced scaffold proteins mimic the function of an antibody as disclosed above and below and can be used either instead of an immunoglobulin-based antibody or in combination with it. In the present invention, said trifunctional antibody can be replaced with said scaffold protein insofar both display the same function in respect of binding to a T cell, binding to a tumor-associated antigen on a tumor cell, and binding via its Fc-portion to an Fc-receptor positive cell, to obtain an intra-operatively salvaged blood containing cell aggregates. In the present invention, said trifunctional antibody and said scaffold protein can be applied alone or in combination.

[0146] Preferably, the antibodies to be used in the present invention are characterized by the additional effects of binding of the Fc receptor-positive cell by binding to the Fc receptor-positive cell via Fey receptors of type I, II and III.

[0147] Preferably, the antibody used according to the invention comprises a binding site in its Fc-portion for Fey receptor type I, II and / or III.

[0148] Preferably, the antibody used according to the invention is able to bind to monocytes, macrophages, dendritic cells, natural killer cells, neutrophils and / or eosinophilic cells being Fey receptor positive cells.

[0149] In the present invention, the tumor associated antigen refers to an antigenic substance produced in tumor cells which is expressed on the surface of a tumor cell. Preferably, the pan-leukocyte antigen is selected from a group consisting of CD1 1 a, CD15, CD18, CD29, CD39, CD45, CD48, CD52, CD55, CD58, CD59, CD82, CD95, CD97, CD122, CD124, CD132, and CDw137, more preferably the pan-leukocyte antigen is CD52.

[0150] Preferably, the T cell surface antigen is selected from a group consisting of CD2, CD3, CD4, CD5, CD6, CD8, CD28, CD40L and CD44, CD80, CD86, more preferably the T cell surface antigen is CD3.

[0151] Preferably, the tumor associated antigen which is selected from the group consisting of: EpCAM, Her2neu, EGFR, CD30, CD38, CD79, CD123, CD133, PD-L1 CD20, CD22, MUC-1 , MUC-16, MUC-17, MUC1 * with changed glycosylation pattern, PSMA, CD33, MCSP, cMet, EphA2, Endosialin, Carboanhydrase IX, IGF- 1 R, FAP-alpha, CD19, BCMA, GD2, CEA, proteoglycans, G250, GC182, GT468, GT512, GD3, GM1 , GM2, GM3, fucosyl-GM1 , globo-H, S1 P, Cer, and Gg3, more preferably the tumor associated antigen is EpCAM or GD2.

[0152] More preferably, said tumor associated antigen the trifunctional antibody or scaffold protein binds to is EpCAM, Her2 / neu, MUC, EGFR, EphA2, GD2 or CD20.

[0153] Preferably, the trifunctional antibody of the present invention is directed against one pan-leukocyte antigen and one tumor-associated antigen, wherein the pan-leukocyte antigen which is selected from a group consisting of CD11 a, CD15, CD18, CD29, CD39, CD45, CD48, CD52, CD55, CD58, CD59, CD82, CD95, CD97, CD122, CD124, CD132, and CDw137, can be combined with any tumor-associated antigen selected from a group consisting of EpCAM, Her2neu, EGFR, CD30, CD20, CD22, MUC1 , MUC1* with changed glycosylation pattern, PSMA, CD33, MCSP, cMet, EphA2, Endosialin, Carboanhydrase, IGF-1 R, FAP- alpha, CD19, GD2, CEA, FR, proteoglycans, G250, GC182, GT468, and GT512.

[0154] Preferably, the trifunctional antibody of the present invention is directed against one pan-leukocyte antigen which is CD52 and one tumor-associated antigen which is EpCAM.

[0155] Preferably, the trifunctional antibody of the present invention is directed against one pan-leukocyte antigen and one tumor-associated antigen wherein more preferably said tumor cell is a carcinoma cell.

[0156] Preferably, the trifunctional antibody of the present invention is directed against one pan-leukocyte antigen which is CD52, one tumor-associated antigen which is EpCAM wherein more preferably said tumor cell is a carcinoma cell. Preferably, the trifunctional antibody of the present invention is directed against one pan-leukocyte antigen, one tumor-associated antigen wherein more preferably said tumor cell is a carcinoma cell, and one Fc- gamma receptor type I.

[0157] Preferably, the trifunctional antibody of the present invention is directed against one pan-leukocyte antigen which is CD52, one tumor-associated antigen which is EpCAM wherein more preferably said tumor cell is a carcinoma cell, and one gamma receptor type which is CD64. Said trifunctional antibody can lead to 3- dimensional cell complexes or aggregates, as illustrated in Figure 1.

[0158] Preferably, the trifunctional antibody of the present invention is directed against one T cell surface antigen and one tumor-associated antigen, wherein the T cell surface antigen which is selected from a group consisting of CD2, CD3, CD4, CD8, CD28, CD40L and CD44, can be combined with any tumor-associated antigen selected from a group consisting of EpCAM, Her2neu, EGFR, CD30, CD20, CD22, MUC1 , MUC1 * with changed glycosylation pattern, PSMA, CD33, MCSP, cMet, EphA2, Endosialin, Carboanhydrase, IGF- 1 R, FAP-alpha, CD19, GD2, CEA, FR, proteoglycans, G250, GC182, GT468, and GT512.

[0159] Preferably, the trifunctional antibody of the present invention is directed against one T cell surface antigen which is CD3 and one tumor-associated antigen which is EpCAM.

[0160] Preferred antibodies are heterologous trifunctional bispecific antibodies, preferably monoclonal, selected from one or more of the following combinations of isotypes:

[0161] • rat-lgG2b / mouse-lgG2a,

[0162] • rat-lgG2b / mouse-lgG2b,

[0163] • rat-lgG2b / mouse-lgG3;

[0164] • rat-lgG2b / human-lgG1 ,

[0165] • rat-lgG2b / human-lgG2

[0166] • rat-lgG2b / human-lgG3 [oriental allotype G3m(st)=binding to protein A], rat-lgG2b / human-lgG4;

[0167] • rat-lgG2b / rat-lgG2c;

[0168] • mouse-lgG2a / human-lgG3 [Caucasian allotypes G3m(b+g)=no binding to protein A, in the following indicated as *]

[0169] • mouse-lgG2a / mouse-[VH-CH1 , VL-CL]-human-lgG1 -[hinge]-human-lgG3*-[CH2-CH3]

[0170] • mouse-lgG2a / rat-[VH-CH1 , VL-CL]-human-lgG1 -[hinge]-human-lgG3*-[CH2-CH3] • mouse-lgG2a / human-[VH-CH1 , VL-CL]-human-lgG1 -[hinge]-human-lgG3*-[CH2-CH3]

[0171] • mouse-[VH-CH1 , VL-CL]-human-lgG1 / rat-[VH-CH1 , VL-CL]-human-lgG1 -[hinge]-human-lgG3*-[CH2- CH3]

[0172] • mouse-[VH-CH1 , VL-CL]-human-lgG4 / rat-[VH-CH1 , VL-CL]-human-lgG4-[hinge]-human-lgG4 [N- terminal region of CH2]-human-lgG3*[C-terminal region of CH2:>aa position 251]-human-lgG3*[CH3]

[0173] • at-lgG2b / mouse-[VH-CH1 , VL-CL]-human-lgG1 -[hinge-CH2-CH3]

[0174] • rat-lgG2b / mouse-[VH-CH1 , VL-CL]-human-lgG2-[hinge-CH2-CH3]

[0175] • rat-lgG2b / mouse-[VH-CH1 , VL-CL]-human-lgG3-[hinge-CH2-CH3, oriental allotype]

[0176] • rat-lgG2b / mouse-[VH-CH1 , VL-CL]-human-lgG4-[hinge-CH2-CH3]

[0177] • human-lgG1 / human-[VH-CH1 , VL-CL]-human-lgG1 -[hinge]-human-lgG3*-[CH2-CH3]

[0178] • human-lgG1 / rat-[VH-CH1 , VL-CL]-human-lgG1 -[hinge]-human-lgG4 [N-terminal region of CH2]- human-lgG3*[C-terminal region of CH2:>aa position 251]-human-lgG3*[CH3]

[0179] • human-lgG1 / mouse-[VH-CH1 , VL-CL]-human-lgG1 -[hinge]-human-lgG4 [N-terminal region of CH2]- human-lgG3*[C-terminal region of CH2:>aa position 251]-human-lgG3*[CH3]

[0180] • human-lgG1 / rat-[VH-CH1 , VL-CL]-human-lgG1 -[hinge]-human-lgG2 [N-terminal region of CH2]- human-lgG3*[C-terminal region of CH2:>aa position 251]-human-lgG3*[CH3] human-lgG1 / mouse-[VH-CH1 , VL-CL]-human-lgG1 -[hinge]-human-lgG2 [N-terminal region of CH2]- human-lgG3*[C-terminal region of CH2:>aa position 251]-human-lgG3*[CH3]

[0181] • human-lgG1 / rat-[VH-CH1 , VL-CL]-human-lgG1 -[hinge]-human-lgG3*-[CH2-CH3]

[0182] • human-lgG1 / mouse-[VH-CH1 , VL-CL]-human-lgG1 -[hinge]-human-lgG3*-[CH2-CH3]

[0183] • human-lgG2 / human-[VH-CH1 , VL-CL]-human-lgG2-[hinge]-human-lgG3*-[CH2-CH3]

[0184] • human-lgG4 / human-[VH-CH1 , VL-CL]-human-lgG4-[hinge]-human-lgG3*-[CH2-CH3]

[0185] • human-lgG4 / human-[VH-CH1 , VL-CL]-human-lgG4-[hinge]-human-lgG4 [N-terminal region of CH2]- human-lgG3*[C-terminal region of CH2:>aa position 251]-human-lgG3*[CH3]

[0186] • mouse-lgG2b / rat-[VH-CH1 , VL-CL]-human-lgG1 -[hinge]-human-lgG3*-[CH2-CH3]

[0187] • mouse-lgG2b / human-[VH-CH1 , VL-CL]-human-lgG1 -[hinge]-human-lgG3*-[CH2-CH3]

[0188] • mouse-lgG2b / mouse-[VH-CH1 , VL-CL]-human-lgG1 -[hinge]-human-lgG3*-[CH2-CH3]

[0189] The trifunctional bispecific antibody with monovalent binding specificities used in one particularly preferred embodiment by the present invention has the following properties: the three arms of said trifunctional antibody respectively comprises the following properties: a) binding to a leukocyte or a tumor-associated antigen on a tumor cell or leukocyte; b) binding to a tumor-associated antigen on a tumor cell; c) binding via its Fc-portion to an Fc-receptor positive cell, wherein preferably the tumor cell in a) are of hematological origin such as leukemias or lymphomas, and b) of epithelial or neuroektodermal origin like such as carcinoma or melanoma or sarkoma, small cell lung cancer or glioma, and wherein the trifunctional bispecific antibody is further preferably selected of a group of antibodies with the following isotype combinations: rat-lgG2b / mouse-lgG2a, rat-lgG2b / mouse-lgG2b, rat-lgG2b / human-lgG1 , mouse-[VH-CH1 ; VL-CL]-human-lgG1 / rat-[VH-CH1 , VL-CL]-human-lgG1 -[hinge]-human-lgG3*- [CH2-CH3]

[0190] [* = Caucasian allotypes G3m(b+g) = no binding to protein A],

[0191] Specifically preferred is an antibody, preferably a trifunctional bispecific antibody and / or a scaffold protein directed against EpCAM and CD52 with the isotype combination rat-lgG2b / mouse-lgG2a. A preferred example for said trifunctional bispecific antibody is anti-CD52 x anti-EpCAM antibody. Preferably said antibody is monoclonal.

[0192] Specifically preferred is an antibody, preferably a trifunctional bispecific antibody and / or a scaffold protein directed against EpCAM and CD3 with the isotype combination rat-lgG2b / mouse-lgG2a. A preferred example for said trifunctional bispecific antibody is anti-CD3 x anti-EpCAM antibody. Preferably said antibody is monoclonal.

[0193] Preferably, the antibodies according to the invention are monoclonal, chimeric, recombinant, synthetic, semi-synthetic, or chemically modified intact antibodies having for example Fv, Fab, scFv, or F (ab)2 fragments.

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

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

[0196] It is further possible to prepare the antibodies described by means of recombinant DNA technology according to techniques obvious to the skilled artisan (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, the so-called bispecific antibodies, can be performed for example using recombinant DNA technology but also by the so-called hybrid hybridoma fusion technique (see for example Milstein et al., Nature 305 (1983), 537). This technique comprises fusing hybridoma cell lines each producing antibodies having one of the desired specificities and identifying and isolating recombinant cell lines producing antibodies having both specificities.

[0198] The problem forming the basis of the invention can be overcome by using in preferred embodiments either trifunctional bispecific or trispecific trifunctional antibodies if they exhibit the properties and effects as described herein. The invention is particularly described by the way of trifunctional bispecific antibodies. However, it is understood that it also covers the following trispecific antibodies exhibiting similar effects. Although above, the terms “antibody” or “scaffold protein” may refer to trifunctional bispecific antibody, it is understood that it can also covers the following trispecific antibodies exhibiting similar effects.

[0199] The preparation of antibodies exhibiting three specificities, so-called trispecific antibodies, also suitable to solve the problem of the invention, may for example be carried out by coupling a third antigen binding site having an additional specificity, e.g. in the form of "single chain variable fragments" (scFv) to one of the IgG heavy chains of a bispecific antibody. Further, recombinant technology can be used, e.g. vector-based methods for protein synthesis or oligonucleotide synthesis.

[0200] Analogously, trispecific F(ab)2 constructs may be prepared by replacing the CH2-CH3 regions of the heavy chain of one specificity of a bispecific antibody by an scFv having a third specificity, while the CH2-CH3 regions of the heavy chain having the other specificity can be removed for example by insertion of a stop codon (at the end of the "hinge" 5 region) into the coding gene, e.g by homologous recombination.

[0201] It is also possible to prepare trispecific scFv constructs wherein three VH-VL regions representing three different specificities are arranged in series.

[0202] Intact bispecific antibodies are composed of two antibody semi-molecules (each having a H and a L immunoglobulin chain) each representing a specificity, and additionally like normal antibodies having a Fc portion performing the well-known effector functions. They are preferably prepared using the quadroma technology. This method of preparation is exemplified in DE-A-44 19 399. For complete disclosure this document is incorporated in its entirety by reference also with respect to a definition of bispecific antibodies. It should be understood that other methods of preparation are also useful if they lead to the intact bispecific antibodies according to the above definition required according to the invention.

[0203] For example, intact bispecific antibodies may be produced in sufficient amounts using a newly developed method of preparation (Lindhofer et al., J. Immunology, 155:219 (1995)). The combination of two bispecific antibodies directed against two different tumor-associated antigens (e.g. c-erb-B2, EpCAM, such as GA- 733-2=C215) on the mammary carcinoma cells minimizes the risk that tumor cells expressing only one of the antigens remain unidentified.

[0204] The terms "associates", "multi-cellular complexes" and "aggregates" are used interchangeably and always define a three-dimensional network between antibodies, scaffold proteins, tumor cells and / or immune cells and / or further tumor cells in order to form cross-linked tumor cells which are removable by filtration or column beads. Said associates are specifically comprised of antibodies, tumor cells and immune cells which are leukocyte e.g.T cells and / or Fc-receptor positive cells.

[0205] Preferably, the EC is contacted with above antibody at room temperature which might range from 19°C to 25°C, preferably about 21 °C.

[0206] Preferably, the purified EC is isolated from said bispecific and / or the multispecific antibody or a combination of multispecific / bispecific and / or a combination of parental antibodies that bind with unwanted nucleated cells, preferably lymphocytes, leukocytes and / or tumor cells.

[0207] Preferably, said isolation is performed through applying a filtration.

[0208] The filtration is preferably a leukocyte reduction or depletion filtration wherein cell aggregates or associates containing tumor cells or tumor cell - leukocyte cell complexes are all or substantially removed while the erythrocytes pass the filter and are collected. The filters might be selected from screen filters with pore sizes of between 16-20 pm to 40 pm which retain the associates formed by the method of the invention and also for instance fibrin strands and clumps of dead cells. Erythrocytes which are about 8 pm in size may pass through the filters.

[0209] Preferably, above cell aggregates may contain one of following combinations: (1 ), antibody and tumor cells; (2) antibody, tumor cells and leukocyte cells; (3) antibody and leukocyte cells; (4) antibody, tumor cells, immune cells and Fc-gamma receptor-positive cells, and (5) antibody, immune cells and Fc-gamma receptor-positive cells. A review describing the cell saver 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.

[0210] An example for a micro-aggregate blood filter in order to receive an erythrocyte containing fraction only is Pall SQ 40S Blood Transfusion Filter.

[0211] Filtration is performed generally for a time period sufficient to remove said tumor cells-comprising cell associates as well as other unwanted nucleated cells such as lymphocytes or leukocytes of various origins and to separate EC from said tumor cell associates and / or other unwanted nucleated cells such as lymphocytes or leukocytes of various origins.

[0212] In a preferred embodiment said concentrate of erythrocytes which is free or substantially free of contaminating tumor cells and other unwanted nucleated cells such as lymphocytes or leukocytes is used without addition of physiological saline.

[0213] Further filtration steps may be used in order to remove residual tumor cell associates and / or other unwanted nucleated cells such as lymphocytes or leukocytes.

[0214] As the underlying filtration technology is different from classical, micron-rated filtration procedures including clogging issues, this advanced tumor cell removal strategy appears to be feasible.

[0215] It is one of the benefits of the invention that the time involved for said removal of tumor cells and / or other unwanted nucleated cells such as lymphocytes or leukocytes from EC is dramatically reduced by the invention to e.g. 5-3 0 min, compared to the depletion of tumor cells by destruction with antibodies or by removal with magnetic beads or cell sorting methods or irradiation.

[0216] Other advantages of the present invention include that tumor cells and / or other unwanted nucleated cells such as lymphocytes or leukocytes from the purified EC are remarkably reduced.

[0217] The EC purified by the method of the invention are - according to a preferred embodiment - then administered into another subject who is the recipient in need of said purified EC, which would not cause or reduce tumor metastases, recurrence of tumor or immunomodulation. Preferably, the recipients who receive the purified EC obtained by the method of the present invention are immuno-compromised or-suppressed recipients. Even in these embodiments, the appearance of tumor or recurrence of tumor can be remarkably suppressed.

[0218] Examples

[0219] Removal of tumor cells from matched allogeneic blood collections

[0220] As allogeneic blood collections can potentially contain tumor cells from donors (Molodysky et al., Asian Pac J Cancer Prev, 22 (3), 641 -649, 2021 ), we tested whether the Catuvab procedure is able to remove tumor cells from matched allogeneic blood collections using a cell saver device.

[0221] To test the Catuvab procedure, a defined amount of EpCAM positive tumor cells was spiked into two blood products - matched for a hypothetical recipient - and collected within the reservoir of a cell saver device.

[0222] In the here described example, two blood collections with a volume of 550ml each, blood group B, Rhesus pos, and Hb 17.6 were applied to the reservoir of a cell saver device (Sorin - XTRA) .

[0223] Subsequently, 500x10e3 SW480 colon carcinoma tumor cells were added (0.5x10e3 tumor cells / ml RPMI medium) via a connector on the top of the reservoir.

[0224] In the meantime, 100pl of the catumaxomab antibody concentrate (1 Opig / 1 OOpil) of the Catuvab kit was diluted within 5.7ml sterile saline. From this dilution, 1.5ml volume was taken - representing ca. 2.5pg antibody - and applied, via a connector on the top of reservoir, into the described blood collection.

[0225] After 30min incubation time, the cell saver was started to produce an erythrocyte concentrate (EC). From the 1 .1 L blood collections an EC of 640ml was generated.

[0226] This EC was subsequently filtrated via gravity by using a Fresenius BioR flex leukocyte depletion filter to remove potentially present cell complexes of lymphocytes and tumor cells induced by catumaxomab.

[0227] The final blood product had a volume of about 610ml, allowing to calculate a dead volume of the Fresenius LDF of about 30ml.

[0228] The analysis for EpCAM positive tumor cells in the reservoir and the final EC blood product is shown in table 1

[0229] Table 1

Claims

Claims1 . An ex vivo method for preparing purified erythrocyte concentrates (EC) or a whole blood product for transfusion, comprising a step of contacting an allogeneic blood collection with a bispecific and / or a multispecific antibody or a combination of parental antibodies in a blood salvage device (BSD).

2. The ex vivo method of claim 1 , wherein said BSD is a cell saver machine or a filtration system.

3. The ex vivo method of claim 1 or 2, wherein said allogeneic blood collection is a mixture of blood collections from two or more different donors.

4. The ex vivo method of any one of claims 1 to 3, wherein the contacting step is performed by incubating said allogeneic blood collection with said bispecific and / or said multispecific antibody or said combination of parental antibodies for a predetermined time period, so that said bispecific and / or the multispecific antibody or the combination of parental antibodies bind with unwanted nucleated cells, preferably lymphocytes, leukocytes and / or tumor cells.

5. The ex vivo method of any one of claims 1 to 4, wherein erythrocyte concentrates (EC) are generated in the BSD by a centrifugation step and / or a filtration step, or wherein erythrocyte concentrates (EC) are generated in the BSD by a filtration step without any centrifugation step.

6. The ex vivo method of claim 5, comprising a further step of washing said bispecific and / or the multispecific antibody or the combination of parental antibodies that bind with unwanted nucleated cells, preferably lymphocytes, leukocytes and / or tumor cells, wherein said washing step is performed before the centrifugation step and the filtration step, or after the centrifugation step and the filtration step, or before the filtration step if no centrifugation step is performed.

7. The ex vivo method of any one of claims 1 to 6, wherein unwated nucleated cells, preferably lymphocytes, leukocytes and / or tumor cells, are removed or their amount is reduced from said EC, more preferably at least 95% of tumor cells being removed and / or at least 80% of leukocytes, lymphocytes being removed.

8. The ex vivo method of any one of claims 1 to 7, wherein the bispecific and / or the multispecific antibody is a trifunctional antibody, wherein the three arms of said trifunctional antibody respectively comprises the following properties: a) binding to a leukocyte or a tumor-associated antigen on a tumor cell or leukocyte; b) binding to a tumor-associated antigen on a tumor cell;c) binding via its Fc-portion to an Fc-receptor positive cell, wherein preferably the tumor cell or the leukocyte in a) are of hematological origin such as leukemias or lymphomas, and b) of epithelial or neuroektodermal origin like such as carcinoma or melanoma or sarkoma, small cell lung cancer or glioma.

9. The ex vivo method of claim 8, wherein the three arms of said trifunctional antibody respectively comprises the following properties: a) binding to a tumor-associated antigen on a tumor cell; b) binding to a tumor-associated antigen on a tumor cell which is different from the antigen in a); c) binding via its Fc-portion to an Fc-receptor positive cell.

10. The ex vivo method of claim 8 or 9, wherein the arm of the trifunctional antibody corresponding to the property a) can bind to a pan-leukocyte antigen, which is selected from a group consisting of CD11 a, CD15, CD18, CD29, CD39, CD45, CD48, CD52, CD55, CD58, CD59, CD82, CD95, CD97, CD122, CD124, CD132, and CDw137, preferably the pan-leukocyte antigen is CD52.11 . The ex vivo method of claim 8 or 9, wherein the arm of the trifunctional antibody corresponding to the property a) can bind to T cell through a T cell surface antigen, and wherein the T cell surface antigen is selected from a group consisting of CD2, CD3, CD4, CD5, CD6, CD8, CD28, CD40L and CD44, CD80, CD86, preferably the T cell surface antigen is CD3.

12. The ex vivo method of any one of claims 8-11 , wherein the arm of the trifunctional antibody corresponding to the property b) can bind to a tumor associated antigen which is selected from the group consisting of: EpCAM, Her2neu, EGFR, CD30, CD38, CD79, CD123, CD133, PD-L1 CD20, CD22, MUC-1 , MUC-16, MUC-17, MUC1 * with changed glycosylation pattern, PSMA, CD33, MCSP, cMet, EphA2, Endosialin, Carboanhydrase IX, IGF-1 R, FAP-alpha, CD19, BCMA, GD2, CEA, proteoglycans, G250, GC182, GT468, GT512, GD3, GM1 , GM2, GM3, fucosyl-GM1 , globo-H, S1 P, Cer, and Gg3, preferably the tumor associated antigen is EpCAM or CD20 or GD2.

13. The ex vivo method of claim 8 or 9, wherein the arms of the trifunctional antibody corresponding to the property a) and b) can bind to a tumor associated antigen which is selected from the group consisting of: EpCAM, Her2neu, EGFR, CD30, CD38, CD123, CD133, PD-L1 CD20, CD22, MUC1 , MUC1 * with changed glycosylation pattern, PSMA, CD33, MCSP, cMet, EphA2, Endosialin, Carboanhydrase IX, IGF-1 R, FAP- alpha, CD19, BCMA, GD2, CEA, FR, proteoglycans, G250, GC182, GT468, GT512, GD3, GM1 , GM2,GM3, fucosyl-GM1 , globo-H, S1 P, Cer, and Gg3, preferably the tumor associated antigen is EpCAM or CD20 or GD2.

14. The ex vivo method of any one of claims 8-13, wherein the trifunctional antibody comprises a binding site in its Fc-portion for Fey receptor type I, II and / or III.

15. The ex vivo method of any one of claims 8-14, wherein the trifunctional antibody can bind to monocytes, macrophages, dendritic cells, natural killer cells and / or activated neutrophils via their Fey receptor type I, II and / or III.

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