Pharmaceutical preparation for treating epstein-barr virus positive patients with a disease associated with reactivation phenomenon

Abstract

An isolated trifunctional bispecific antibody for use in a method of treating a patient suffering from a disease and / or disorder associated with reactivation of Epstein-Barr virus (EBV) in at least B cells and potentially other susceptible cells, such as susceptible epithelial cells, the method comprising: providing an autologous cell preparation of enriched B cells of the patient; incubating the enriched B cells with a trifunctional bispecific antibody for a period of time sufficient to establish a physical interaction between the trifunctional bispecific antibody and the enriched B cells to obtain an incubation mixture; transferring the incubation mixture obtained after incubation into the same patient, wherein the trifunctional bispecific antibody comprises: (a) a first binding arm binding to B cells via a B cell surface antigen; (b) a second binding arm binding to T cells via a T cell surface antigen; (c) an Fc part binding to Fc receptor positive cells. The invention also discloses a pharmaceutical composition comprising the isolated trifunctional bispecific antibody for use in treating a patient suffering from a disease and / or disorder associated with reactivation of EBV in B cells, and an ex vivo method for the preparation of the pharmaceutical composition.

Description

TECHNICAL FIELD

[0001] The present application relates to a trifunctional bispecific antibody for use in a method for treating a patient suffering from a disease and / or a disorder associated with the reactivation of Epstein-Barr virus (EBV) in at least B cells and potentially other susceptible cells such as susceptible epithelial cells, to a pharmaceutical composition comprising said antibody for said use, and to an ex vivo method for preparing said pharmaceutical composition for said use. BACKGROUND

[0002] Epstein-Barr virus (EBV) is a double-stranded DNA herpes virus that causes life-long infection in a high proportion (>90%) of the world population. Primary infection usually occurs during childhood and is mostly asymptomatic. In developed countries, these infections are often delayed until adolescence, reaching 50% of cases, which in some individuals can produce severe symptoms that last for many years.

[0003] Up to half of these delayed primary infections are symptomatic, presenting as acute infectious mononucleosis (AIM) or glandular fever, manifested as fever, fatigue, malaise, pharyngitis and lymphadenopathy.

[0004] During the incubation period, the cycle of infection, lytic replication and reinfection initially takes place without interference from cytotoxic CD8 + T cells, as it takes time to raise an adaptive immune response. As a result, the number of potentially infected memory B cells can rise to half, or even more, of the peripheral memory B cell compartment during AIM (Hochberg et al., J. Virol., 78:5194, 2004).

[0005] The difference between asymptomatic primary EBV infection and AIM is thought to be the higher number of EBV-infected B cells in AIM, the symptoms of which are due to the massive destruction of EBV-infected B-cells by cytotoxic CD8 + T cells (Hadinoto et al., Blood, 111 :1420, 2008).

[0006] EBV is the first human DNA virus identified as having oncogenic potential. It has been found to be associated with various human malignancies, such as Burkitt's lymphoma (BL), undifferentiated nasopharyngeal carcinoma (NPC), salivary gland tumors or Hodgkin's disease (HD). But the list of diseases associated with EBV is even still increasing.

[0007] Thus, there is an increasing discussion of EBV as a potential trigger of post-reactivation immunopathologies and related diseases.

[0008] This type of immune disorder may be caused by the uncontrolled repeated activation of the EBV lysis cycle (reactivation phenomenon), which is the opposite of the usually stable EBV latency found in healthy EBV-infected individuals.

[0009] EBV infects almost exclusively B cells via the CD21 surface molecule. Therefore, we applied in situ detection of EBV mRNA within infected cells to estimate the percentage of infected B cells at a given time point (blood sample collection). In healthy EBV-infected individuals, the frequency of infected B cells ranges from 1 to 50 per 10e6 B cells (Cohen, NEJM, 2000). For example, a frequency a thousandfold higher than normal may provide a strong indication of acute reactivation.

[0010] In 2003, Michael Pender hypothesized that EBV infection of autoreactive B cells (up to 20% of B cells) could be the cause of inflammation in specific tissues, depending on the specificity of the B cell receptors of the infected autoreactive B cells (Pender, Trends Immunol., 24:584, 2003).

[0011] Therefore, chronic inflammation can be induced by EBV infection and activated autoreactive B cells in a variety of tissues, particularly in genetically susceptible older adults (if they have Alzheimer's and Parkinson's diseases) as a possible mechanism, and may thus lead to a variety of diseases / symptoms of unknown origin. Examples of such diseases / symptoms include, for example, Alzheimer's disease (Licastro et al., Oncosience, 3:135-142, 2016), Parkinson's disease (Woulfe J., Neurol. Neuroimmunol. Neuroinflamm. 2016), chronic fatigue syndrome, recurrent infections, sleep disorders, night sweats, lymphadenopathy, chronic cystitis, chronic prostatitis, chronic ductal inflammation, acne-like dermatitis, hair loss, poor attention, and memory problems.

[0012] Over the past two decades, numerous publications have also provided data on EBV's involvement in a variety of autoimmune diseases, such as rheumatoid arthritis / Sjögren's syndrome. Syndrome; Multiple sclerosis; Systemic lupus erythematosus; Type 1 diabetes; Crohn's disease / chronic colitis, psoriasis, vitiligo, Hashimoto's thyroiditis, alopecia areata / generalized hair loss, and myasthenia gravis (Pender, Autoimmune Diseases, 2012).

[0013] Rheumatoid arthritis (RA) / Sjogren's syndrome

[0014] One of the earliest direct evidence that EBV can be involved in the pathogenesis of RA was the study of Takei et al. starting in 1997. The authors detected EBV by in situ hybridization for EBER-1 in 23.5% of synovial lining cell samples (n=34). But none of 20 cases of osteoarthritis and 1 case of psoriatic arthritis used as controls were positive (p<0.05).

[0015] The study of Blaschke et al. (2000) generally confirmed the results of Takei et al. because 30% of RA patients (n=55) harbored EBV-DNA in synovial fluid cells, compared to 16% of controls (p=0.02). In addition, the group found a two-fold increase in anti-EBNA-1 antibodies compared to healthy controls (p=0.029). Interestingly, 24% of RA patients had serological evidence of reactivated EBV infection, while none of the controls did (p=0.028).

[0016] Further studies were done by Balandraud et al. in 2003, who found that EBV DNA load was 10-fold higher in RA patients than in normal controls in a study of 84 RA patients and 69 normal controls using real-time qPCR. EBV load was stable over time and not affected by anti-rheumatic drugs or HLA-DR that ameliorate the disease. Thus, in patients with RA, EBV, which is highly recognized by antibodies but never eliminated, is an ideal candidate to cause a chronic immune complex disease and anti-EBV antibody responses should be considered as one of the chronic autoantibody responses most relevant to the development of RA (Van Boekel et al., Arthritis Res., 4:87, 2002). A recent review article summarizes the field (Fust, Eu J MI, 4:267, 2011).

[0017] Multiple sclerosis (MS)

[0018] The involvement of EBV in the pathogenesis of MS has also been intensely discussed. The following findings in MS patients support this hypothesis:

[0019] a) Accumulation of EBV-infected B cells and plasma cells in the brain (Serafini et al., J. Exp. Med, 2007),

[0020] b) Elevated levels of anti-EBV antibodies in serum and cerebrospinal fluid (CSF) (Jaquiery et al., Eur. J. Immunol., 2010),

[0021] c) changes in EBV-specific CD8 T cell immunity (Jaquiery et al., 2010; Pender, J. Neurol. Neurosurg. Psychiatry, 2009) and

[0022] d) increased amounts of EBV in saliva of MS patients compared to controls (Yea et al., Neurology, 2013).

[0023] Currently, the following main mechanisms are discussed to explain these findings and the possible role of EBV in the pathogenesis of MS:

[0024] (i) cross-reactivity (mimicry) between the CNS and EBV antigens,

[0025] (ii) impaired control of EBV infection with reactivation phenomena

[0026] (iii) EBV-specific T cells damage bystanders within the brain and

[0027] (iv) EBV infection of autoreactive B cells, which can provide a costimulatory signal to autoreactive T cells in the brain.

[0028] Type 1 and 2 diabetes

[0029] In the past two decades, the relationship between the onset of type 1 diabetes (T1D) and viral infections has been increasingly discussed. Viruses can be involved in the pathogenesis of T1D in multiple ways. In the case of virus-induced autoimmunity, EBV has been discussed in addition to other viruses such as mumps virus, coxsackie virus, rubella virus and cytomegalovirus (Jun et al., Diabetes Metab. Res. Rev., 2003). Another cause for the development of diabetes (type 1 and 2) can be chronic systemic inflammation. In this context, reactivation of HHV-6 and EBV has been discussed. For example, a recent study by Heaseker et al. (2013) found that high antibody titers of HHV-6 and EBV are associated with diabetes.

[0030] In summary, the existing data suggest that a weak control of existing EBV infection together with genetic factors and other accessory factors (such as low vitamin D levels, other infections) or an unbalanced immune response against EBV due to a relatively late infection (e.g. in adolescents or adults) can trigger a variety of autoimmune disorders.

[0031] So far, there is no therapeutic drug for diseases associated with reactivation of EBV, and there is a need to provide a drug for treating patients with EBV reactivation in B cells.

[0032] It is therefore a first object of the present application to provide a medicament for the treatment of a patient suffering from reactivation of EBV in B cells, in particular for the amelioration or treatment of a disease associated with reactivation of EBV in B cells. To achieve this object, a trifunctional bispecific antibody is used, which is characterized in that the antibody has the following properties: (a) binding to B cells via a B cell surface antigen; (b) binding to T cells via a T cell surface antigen; (c) binding to Fc receptor positive cells via its Fc part.

[0033] It is a second object of the present application to provide a pharmaceutical composition for the treatment of a patient suffering from reactivation of EBV in B cells. To achieve this object, a pharmaceutical preparation for the treatment of a patient suffering from reactivation of EBV in B cells comprising the antibody and optionally a pharmaceutically acceptable carrier and / or excipient is provided.

[0034] It is a third object of the present application to provide an ex vivo method for the preparation of the pharmaceutical composition for the method of treating reactivation of EBV in B cells. To achieve this object, the ex vivo method comprises an incubation step comprising contacting autologous B cells of a patient suffering from reactivation of EBV in B cells with the trifunctional bispecific antibody for a time period sufficient to establish a physical interaction between the trifunctional bispecific antibody and the B cells. SUMMARY

[0035] The inventors found that contacting a trifunctional bispecific antibody and an enriched autologous B cell preparation containing EBV infected B cells ex vivo for a time period sufficient to establish a physical interaction between the trifunctional bispecific antibody and the EBV infected B cells and then retransferring the treated autologous cells into the same patient induces a retargeted killing of EBV infected B cells by T cells and accessory cells. Example 1 shows the retargeted killing of enriched autologous B cell populations. As a result, EBV derived antigens will be phagocytosed by Fc-receptor positive professional antigen presenting cells (APC) such as dendritic cells, Langerhans cells of the skin or macrophages, monocytes, natural killer cells and / or activated neutrophils. Eventually, these ingested EBV derived antigens will be processed and re-presented by the professional APCs (antigen presenting cells) leading to a polyclonal humoral and cellular immune response against the patient's EBV Figure 1 ). As a result, the previously existing but potentially weak or incomplete immune response against EBV will be enhanced and the patient will be enabled to better control EBV by preventing or reducing its reactivation.

[0036] Further aspects and embodiments of the present application are disclosed in the dependent claims and can be derived from the following description and examples, without being limited thereto. Attached Figure Description

[0037] The accompanying drawings illustrate embodiments of the invention and convey a further understanding thereof. Together with the textual description, they serve as an explanation of the concepts and principles of the invention. Other embodiments and further advantages can be derived from the drawings.

[0038] Figure 1 The therapeutic vaccination mechanism involves a trifunctional bispecific antibody that can bind to B cells, T cells, and FcY receptor-positive helper cells, and is used to treat patients with EBV reactivation in B cells and related patient diseases and symptoms.

[0039] Figure 2 Trifunctional bispecific antibody-mediated killing of enriched autologous B cells in vitro.

[0040] Figure 3 In situ hybridization of enriched B cells from patients in Example 2 was performed using the EBV-EBER 1+2 probe.

[0041] Figure 4 In situ hybridization of enriched B cells from patients in Example 3 was performed using the EBV-EBER 1+2 probe.

[0042] Figure 5 In situ hybridization was performed on the enriched B cells from the patient in Example 4 using the EBV-EBER 1+2 probe.

[0043] Figure 6 In situ hybridization of enriched B cells from patients in Example 5 was performed using the EBV-EBER 1+2 probe.

[0044] Figure 7 In situ hybridization of enriched B cells from patients in Example 6 was performed using the EBV-EBER 1+2 probe.

[0045] Figure 8 In situ hybridization of enriched B cells from patients in Example 7 was performed using the EBV-EBER 1+2 probe.

[0046] Figure 9 In situ hybridization of enriched B cells from patients in Example 7 was performed using the EBV-EBER 1+2 probe.

[0047] Figure 10 In situ hybridization of enriched B cells from patients in Example 8 was performed using the EBV-EBER 1+2 probe.

[0048] Figure 11: In situ hybridization of enriched B cells from patient in Example 9 using EBV-EBER 1+2 probes.

[0049] Figure 12 : In situ hybridization of enriched B cells from patient in Example 9 using EBV-EBER 1+2 probes.

[0050] Figure 13 : In situ hybridization of enriched B cells from patient in Example 10 using EBV-EBER 1+2 probes.

[0051] Figure 14 : In situ hybridization of enriched B cells from patient in Example 10 using EBV-EBER 1+2 probes.

[0052] Figure 15 : In situ hybridization of enriched B cells from patient in Example 11 using EBV-EBER 1+2 probes.

[0053] Figure 16 : In situ hybridization of enriched B cells from patient in Example 11 using EBV-EBER 1+2 probes. DETAILED DESCRIPTION

[0055] Definitions

[0056] While the application is susceptible to various modifications and alternative forms, specific embodiments thereof have been shown by way of example in the drawings and are described in detail herein. It should be understood, however, that it is not intended to limit the application to the particular embodiments described. On the contrary, it is intended to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the application as defined by the appended claims.

[0057] Hereinafter, elements of the present application will be described. These elements are listed together with specific embodiments, however, it should be understood that they can be combined in any manner and in any number to form further embodiments. The various described embodiments and preferred embodiments should not be construed as limiting the application to only the explicitly described embodiments. The description should be understood to support and encompass various embodiments that combine the explicitly described embodiments with any number of the disclosed and / or preferred elements. Furthermore, unless the context indicates otherwise, it should be considered that any permutation and combination of all described elements in this application are disclosed by the description of this application.

[0058] Throughout the specification and the subsequent claims, unless the context requires otherwise, the term "comprise" and variations such as "comprises" and "comprising" will be understood to imply the inclusion of a stated member, integer or step, but not the exclusion of any other unstated member, integer or step. The term "consisting of" is a specific implementation of the term "comprising", wherein any other unstated member, integer or step is excluded. In the context of the present application, the term "comprising" encompasses the term "consisting of". Thus, the term "comprising" encompasses "including" as well as "consisting of", e.g. a composition "comprising" X can consist exclusively of X or can include something additional, e.g. X + Y.

[0059] The terms "a" and "an" and "the" and similar referents in the context of describing the application (especially in the context of the following claims) are to be interpreted in an inclusive rather than an exclusive sense, that is, in the sense of "comprising," unless otherwise noted in context. Recitation of numerical ranges by endpoints is merely intended to serve as a shorthand for referring individually to each number falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. No language in the specification should be construed as indicating any non-claimed element as essential.

[0060] The word "substantially" does not exclude "completely," e.g. a composition "substantially free from" Y can be completely free from Y. The word "substantially" can be omitted from the definition of the application where necessary.

[0061] Unless otherwise indicated herein, all values given in the present disclosure are to be understood as supplemented by the word "about".

[0062] A first aspect of the presently disclosed invention is an isolated trifunctional bispecific antibody for use in a method of treating a patient suffering from a disease and / or disorder associated with reactivation of Epstein-Barr virus (EBV) in at least B cells and potentially other susceptible cells, such as susceptible epithelial cells, the method comprising: providing an autologous cell preparation of enriched B cells of the patient; incubating the enriched B cells with a trifunctional bispecific antibody for a period of time sufficient to establish a physical interaction between the trifunctional bispecific antibody and the enriched B cells to obtain an incubation mixture; transferring the incubation mixture obtained after incubation into the same patient, wherein the trifunctional bispecific antibody comprises: (a) a first binding arm binding to B cells via a B cell surface antigen; (b) a second binding arm binding to T cells via a T cell surface antigen; (c) a Fc part binding to Fc receptor positive cells, and wherein the disease and / or disorder associated with reactivation of EBV in B cells is selected from one or more autoimmune diseases consisting of rheumatoid arthritis, Hashimoto's disease, type 1 diabetes, and multiple sclerosis, or wherein the disease and / or disorder associated with reactivation of EBV in B cells is selected from one or more chronic inflammatory diseases consisting of chronic prostatitis, chronic cystitis, chronic hepatitis (non-alcoholic), irritable bowel syndrome (IBS), chronic neuroinflammation, and metabolic syndrome; or wherein the disease and / or disorder associated with reactivation of EBV in B cells is selected from one or more diseases and disorders consisting of Sjogren's syndrome, myasthenia gravis, Crohn's disease, vitiligo, type 2 diabetes, chronic inflammation of the milk ducts, lichen simplex, chronic pancreatitis, chronic bronchitis and chronic chimney symptoms, chronic fatigue syndrome, chronic dry cough, chronic rhinitis, night sweats, sleep disorders; insomnia, polyneuropathic pain, edema (e.g. in fingers, toes and face), endometriosis, ovarian cysts, irregular menstruation, hair loss, memory problems, dry eyes and dry mouth, migraine, common hair loss, acne, weight gain, poor concentration, insulin resistance, diarrhea, and chronic herpes zoster.

[0063] Preferably, the antibody used according to the present invention further comprises an autologous cell preparation of peripheral blood mononuclear cells (PBMCs) of the same patient, wherein the PBMCs are added to the incubation mixture of the enriched B cells and the trifunctional bispecific antibody for a period of time sufficient to establish a physical interaction between the PBMCs and the trifunctional bispecific antibody.

[0064] The PBMCs and autologous B cells can be mixed and incubated with the antibody simultaneously or consecutively.

[0065] The incubation time for establishing a physical interaction between the trifunctional bispecific antibody and the B cells can be 1 min to 60 min, preferably 1 min to 20 min, even more preferably 5 min to 20 min, such as 8 min to 15 min, e.g. 8, 9, 10, 11, 12, 13, 14 or 15 min.

[0066] The incubation time for establishing a physical interaction between the trifunctional bispecific antibody and the PBMCs can be 1 min to 60 min, preferably 1 min to 20 min, even more preferably 5 min to 20 min, such as 8 min to 15 min, or 10 min to 15 min, e.g. 8, 9, 10, 11, 12, 13, 14 or 15 min.

[0067] The inventors found by performing flow cytometry measurements that an incubation time of 10-15 min leads to an excellent binding affinity between the antibody and the B cells or between the antibody and the PBMCs. When the incubation time is less than 10 min, the binding affinity is acceptable, but still not optimal. When the incubation time exceeds 15 min, the binding affinity does not improve significantly anymore.

[0068] According to the present application, the antibody is preferably administered in an amount of 0.1 - 100 μg, more preferably in an amount of 0.4 - 50 μg, further preferably in an amount of 0.6 - 20 μg, even further preferably in an amount of 0.8 - 10 μg.

[0069] In this respect, the trifunctional bispecific antibody used according to the present application can be produced in mice, rats, goats, sheep, rabbits, horses, donkeys, pigs or other animals which can be immunized and which are suitable for the production of antibodies. Preferably, the antibody used according to the present application is produced in mice or rats.

[0070] The antibody used according to the present application can also be produced in transgenic animals. In this respect, the transgenic animal can be a mammal, a bird or a fish.

[0071] The antibody used according to the present application can also be produced in cells which can secrete antibodies. Such cells include, but are not limited to, CHO, 293, COS or NSO cells.

[0072] The term "antibody" encompasses various forms of antibodies, preferably monoclonal antibodies, including but not limited to intact antibodies, antibody fragments, human antibodies, chimeric antibodies, humanized antibodies and genetically engineered antibodies (variant or mutant antibodies), as long as they retain the features according to the present application. Preferred are human or humanized monoclonal antibodies and recombinant antibodies, in particular recombinant monoclonal antibodies. Thus, the antibody according to the present application is preferably a monoclonal antibody. Furthermore, it is also preferred that the antibody is a multichain antibody, i.e. an antibody comprising more than one chain, and thus differs from a single chain antibody.

[0073] As used herein, the term "human antibody" is intended to include antibodies having variable and constant regions derived from human immunoglobulin sequences. Human antibodies are well known in the art (van Dijk, M. A., and van de Winkel, J. G., Curr. Opin. Chem. Biol. 5 (2001) 368-374). Human antibodies can also be produced in transgenic animals (e.g., mice) that, upon immunization, are capable of producing intact repertoires or selected specific human antibodies in the absence of endogenous immunoglobulin production. Transfer of the human germ-line immunoglobulin gene array in such germ-line mutant mice will result in the production of human antibodies upon antigen challenge (see, e.g., Jakobovits, A. et al., Proc. Natl. Acad. Sci. USA 90 (1993) 2551-2555; Jakobovits, A. et al., Nature 362 (1993) 255-258; Bruggemann, M. et al., Year Immunol. 7 (1993) 3340). Human antibodies can also be produced in phage display libraries (Hoogenboom, H. R., and Winter, G., J. Mol. Biol. 227 (1992) 381-388; Marks, J. D., et al., J. Mol. Biol. 222 (1991) 581-597). The techniques of Cole et al. and Boerner et al. are also used to produce human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); and Boerner, P., et al., J. Immunol. 147 (1991) 86-95). The term "human antibody", as used herein, also includes such modified antibodies, e.g., in the variable region, to generate properties according to the present application.

[0074] As used herein, the term "recombinant antibody" is intended to include all antibodies that do not occur in nature, in particular antibodies that are prepared, expressed, produced, or isolated by recombinant means, such as antibodies isolated from a host cell, e.g., a CHO cell, or an animal (e.g., a mouse) or antibodies expressed using a recombinant expression vector transfected into a host cell. Such recombinant antibodies have variable and constant regions that occur in a rearranged form, in contrast to naturally occurring antibodies.

[0075] As used herein, a "trifunctional" antibody is to be understood in the context of the present application as a particular class of bispecific antibodies which recruit B cells and T cells and at the same time also recruit Fc receptor positive cells. In the context of the present application, the trifunctional bispecific antibodies bind via their Fc part to Fc receptor positive cells.

[0076] As used herein, the term "Fc part" refers to a sequence derived from a part of an immunoglobulin heavy chain which starts with the hinge region just upstream of the papain cleavage site and ends with the C-terminus of the immunoglobulin heavy chain. Thus, the "Fc part" can be the complete Fc region or a part thereof (e.g. a domain). Preferably, the "Fc part" mediates the complete functionality of the complete Fc region, e.g. including Fc receptor binding. Thus, the antibodies used according to the present application preferably comprise a complete Fc region, wherein the complete Fc region comprises at least a hinge domain, a CH2 domain and a CH3 domain. The Fc part can also comprise one or more amino acid insertions, deletions or substitutions relative to a naturally occurring Fc region. For example, at least one of the hinge domain, the CH2 domain or the CH3 domain (or a part thereof) can be deleted. For example, the Fc part can comprise or consist of (i) a hinge domain (or a part thereof) fused to a CH2 domain (or a part thereof), (ii) a hinge domain (or a part thereof) fused to a CH3 domain, (iii) a CH2 domain (or a part thereof) fused to a CH3 domain (or a part thereof), (iv) a hinge domain (or a part thereof), (v) a CH2 domain (or a part thereof), or (vi) a CH3 domain or a part thereof.

[0077] Preferably, the trifunctional bispecific antibodies used according to the present application comprise one or more binding sites for Fc receptors in their Fc part.

[0078] More preferably, the trifunctional bispecific antibodies comprise one or more binding sites for Fcy receptors type I, Ila and / or III in their Fc part.

[0079] Further preferably, the trifunctional bispecific antibodies comprise one binding site for Fcy receptors type I, Ila and / or III in their Fc part.

[0080] Cells comprising Fcy receptors type I, Ila and / or III include, but are not limited to, monocytes, macrophages, dendritic cells, natural killer cells and / or activated neutrophilic granulocytes. Preferably, the trifunctional bispecific antibodies are capable of binding monocytes, macrophages, dendritic cells, natural killer cells and / or activated neutrophilic granulocytes via their Fcy receptors type I, Ila and / or III.

[0081] The trifunctional bispecific antibodies used according to the present application can exhibit in their Fc part one of the following isotype combinations: rat-IgG2b / mouse-IgG2a, rat-IgG2b / mouse-IgG2b, rat-IgG2b / human-IgG1, human-IgG1 / human-IgG1-[hinge]-human IgG3*-[CH2-CH3], wherein * = Caucasian allotype G3m(b+g) = no binding to Protein A; mouse-[VH-CH1, VL-CL]-human-IgG1 / rat-[VH-CH1, VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3], wherein * = Caucasian allotype G3m(b+g) = no binding to Protein A; mouse-[VH-VL]-human-[CH1-CL]-human-IgG1 / rat-[VH-CH1, VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3], wherein * = Caucasian allotype G3m(b+g) = no binding to Protein A.

[0082] Preferably, the trifunctional bispecific antibody is a rat / mouse bispecific antibody and exhibits in its Fc part the isotype combination rat-IgG2b / mouse-IgG2a.

[0083] As used herein, the term "bispecific" refers to the ability to bind to two different epitopes, i.e. on a T cell surface antigen and on a B cell antigen. Furthermore, a single "specificity" can refer to one, two, three or more identical paratopes in a single antibody (the actual number of paratopes in a single antibody molecule is referred to as the "valency"). For example, a single native IgG antibody is monospecific and bivalent, as it has two identical paratopes. As used herein, a "paratope" refers to the epitope binding site of an antibody. Thus, the term "bispecific antibody" refers to an antibody with two different paratopes and the ability to bind to two different epitopes.

[0084] Preferably, the bispecific antibody according to the application can comprise four paratopes, wherein each two paratopes are identical (i.e. have the same specificity), and thus the antibody is bispecific and tetravalent (two identical paratopes for each of the two specificities). Thus, the "two specificities" can be realized by two, three, four, five, six, eight, ten, twelve or more paratopes, as long as they refer to only two specificities. Most preferably, the bispecific antibody comprises one single paratope for each specificity, i.e. the bispecific antibody comprises two paratopes in total. The bispecific antibody further preferably comprises two identical paratopes for each of the two specificities, i.e. the bispecific antibody comprises four paratopes in total. Preferably, the bispecific antibody comprises three (identical) paratopes for each of the two specificities, i.e. the bispecific antibody comprises six paratopes in total.

[0085] As used herein, the term "antigen" refers to any structural substance that serves as a target for receptors of an adaptive immune response, in particular for antibodies, T cell receptors and / or B cell receptors. An "epitope", also known as an "antigenic determinant", is a part (or fragment) of an antigen that is recognized by the immune system, in particular by antibodies, T cell receptors and / or B cell receptors. Thus, an antigen has at least one epitope, i.e. a single antigen has one or more epitopes. An antigen can be (i) a peptide, polypeptide or protein, (ii) a polysaccharide, (iii) a lipid, (iv) a lipoprotein or lipopeptide, (v) a glycolipid, (vi) a nucleic acid, or (vii) a small molecule drug or toxin. Thus, an antigen can be a peptide, a protein, a polysaccharide, a lipid, a combination thereof including lipoproteins and glycolipids, a nucleic acid (e.g. DNA, siRNA, shRNA, antisense oligonucleotide, decoy DNA, plasmid) or a small molecule drug (e.g. cyclosporin A, paclitaxel, doxorubicin, methotrexate, 5-aminolevulinic acid) or any combination thereof. Preferably, the antigen is selected from (i) a peptide, polypeptide or protein, (ii) a polysaccharide, (iii) a lipid, (iv) a lipoprotein or lipopeptide, and (v) a glycolipid; more preferably, the antigen is a peptide, polypeptide or protein.

[0086] As used herein, "B cell surface antigen" means a B cell surface associated antigen or a B cell surface specific antigen and "T cell surface antigen" means a T cell surface associated antigen or a T cell specific antigen. In the context of the present application, the B cell surface antigen or the T cell surface antigen can be a cluster of differentiation (CD) molecule. CD molecules are markers that can be used to identify cells on the surface of white blood cells. The bispecific antibody used according to the present application can bind to a B cell via a B cell surface antigen selected from the group consisting of CD19, CD20, CD21, CD22, CD23, CD24, CD37, CD38, CD72, CD75, CD78, CD79 and CD80. This means that the antibody used according to the present application preferably comprises a paratope that can recognize and bind to an epitope of a B cell surface antigen selected from the group consisting of CD19, CD20, CD21, CD22, CD23, CD24, CD37, CD38, CD72, CD75, CD78, CD79 and CD80. This specificity preferably facilitates the recruitment of B cells.

[0087] Preferably, the B cell surface antigen is CD20. This means that the antibody used according to the present application further preferably comprises a paratope that can recognize and bind to an epitope of CD20.

[0088] The bispecific antibody used according to the present application can bind to a T cell via a T cell surface antigen selected from the group consisting of CD2, CD3, CD4, CD8, CD28, CD40L and CD44. This means that the antibody used according to the present application preferably comprises a paratope that can recognize and bind to an epitope of a T cell surface antigen selected from the group consisting of CD2, CD3, CD4, CD8, CD28, CD40L and CD44. This specificity preferably facilitates the recruitment of T cells.

[0089] Preferably, the T cell surface antigen is CD3. This means that the antibody used according to the present application further preferably comprises a paratope that can recognize and bind to an epitope of CD3.

[0090] The bispecific antibody used according to the present application can: (1) bind via its first paratope to an epitope of a B cell surface antigen selected from the group consisting of CD19, CD20, CD21, CD22, CD23, CD24, CD37, CD38, CD72, CD75, CD78, CD79 and CD80, preferably CD20; (2) simultaneously bind via its second paratope to an epitope of a T cell surface antigen selected from the group consisting of CD2, CD3, CD4, CD8, CD28, CD40L and CD44, preferably CD3.

[0091] The bispecific antibody used according to the present application can comprise one paratope against CD3 and one paratope against CD20 (anti-CD3 x anti-CD20). The antibody can comprise one paratope against CD3 and one paratope against CD19 (anti-CD3 x anti-CD19). The antibody can comprise one paratope against CD3 and one paratope against CD22 (anti-CD3 x anti-CD22). The antibody can comprise one paratope against CD3 and one paratope against CD38 (anti-CD3 x anti-CD38). This means that the antibody used according to the present application is preferably selected from the group consisting of anti-CD3 x anti-CD20, anti-CD3 x anti-CD19, anti-CD3 x anti-CD22, anti-CD3 x anti-CD38 bispecific antibodies.

[0092] More preferably, the bispecific antibody used according to the present application comprises an anti-CD3 x anti-CD20 bispecific antibody.

[0093] Thus, the trifunctional bispecific antibody used according to the present application can comprise: (1) one paratope which can recognize and bind to an epitope of a B cell surface antigen selected from the group consisting of CD19, CD20, CD21, CD22, CD23, CD24, CD37, CD38, CD72, CD75, CD78, CD79 and CD80, preferably CD20; (2) one paratope which can recognize and bind to an epitope of a T cell surface antigen selected from the group consisting of CD2, CD3, CD4, CD8, CD28, CD40L and CD44, preferably CD3; (3) one Fc portion which can bind to Fc receptor positive cells, preferably one Fc portion comprising binding sites for Fc gamma receptors type I, Ila and / or III.

[0094] Preferably, the trifunctional bispecific antibody used according to the present application comprises: (1) one paratope which can recognize and bind to an epitope of a B cell surface antigen selected from the group consisting of CD19, CD20, CD21, CD22, CD23, CD24, CD37, CD38, CD72, CD75, CD78, CD79 and CD80, preferably CD20; (2) one paratope which can recognize and bind to an epitope of a T cell surface antigen selected from the group consisting of CD2, CD3, CD4, CD8, CD28, CD40L and CD44, preferably CD3; (3) one Fc portion which can bind to Fc receptor positive cells, preferably one Fc portion which contains binding sites for Fc gamma receptors type I, Ha and / or III, and more preferably the Fc portion contains a combination of isotypes selected from the group consisting of rat-IgG2b / mouse-IgG2a, rat-IgG2b / mouse-IgG2b, rat-IgG2b / human-IgG1, human-IgG1 / human-IgG1-[hinge]-human IgG3*-[CH2-CH3], wherein * = Caucasian allotype G3m(b+g) = no binding to Protein A, or mouse-[VH-CH1, VL-CL]-human-IgG1 / rat-[VH-CH1, VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3], wherein * = Caucasian allotype G3m(b+g) = no binding to Protein A, and even more preferably the Fc portion contains a combination of isotypes of rat-IgG2b / mouse-IgG2a.

[0095] The trifunctional bispecific antibody used according to the present application can be selected from the group consisting of anti-CD3 x anti-CD20, anti-CD3 x anti-CD19, anti-CD3 x anti-CD22, anti-CD3 x anti-CD38 bispecific antibodies, preferably having a combination of isotypes selected from the group consisting of rat-IgG2b / mouse-IgG2a, rat-IgG2b / mouse-IgG2b, rat-IgG2b / human-IgG1, human-IgG1 / human-IgG1-[hinge]-human IgG3*-[CH2-CH3], wherein * = Caucasian allotype G3m(b+g) = no binding to Protein A; or mouse-[VH-CH1, VL-CL]-human-IgG1 / rat-[VH-CH1, VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3], wherein * = Caucasian allotype G3m(b+g) = no binding to Protein A, and even more preferably having a combination of isotypes of rat-IgG2b / mouse-IgG2a.

[0096] In the present application, the two paratope and Fc portion isotype combinations disclosed above can be combined in any combination, if appropriate. The trifunctional bispecific antibodies used according to the present application can be anti-CD3 x anti-CD20 with the following isotype combinations: rat-IgG2b / mouse-IgG2a, human-IgG1 / human-IgG1-[hinge]-human IgG3*-[CH2-CH3], wherein * = Caucasian allotype G3m(b+g) = no binding to Protein A; mouse-[VH-CH1, VL-CL]-human-IgG1 / rat-[VH-CH1, VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3], wherein * = Caucasian allotype G3m(b+g) = no binding to Protein A or mouse-[VH-VL]-human- [CH1-CL]-human-IgG1 / rat-[VH-CH1, VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3], wherein * = Caucasian allotype G3m(b+g) = no binding to Protein A. Further combinations of the two paratope and isotype combinations of the Fc portion can be obtained by the person of ordinary skill without inventive capacity; the further combinations can be useful depending on the different experiments or practical circumstances. Although the following examples have been carried out with antibodies having the above combinations, the present application can also be carried out with any other trifunctional bispecific antibody described herein to obtain the same effect.

[0097] Preferably, in the context of the present application, the bispecific antibody can be any bispecific antibody format, for example as described in Spiess C., Zhai Q. and Carter P.J. (2015) Molecular Immunology 67:95-106. For example, the bispecific antibody can be a whole antibody, for example a whole IgG-like molecule, or a fragment thereof that is not a whole antibody but retains the properties of an antibody. These can be small recombinant formats, for example as tandem single chain variable fragment molecules, diabodies, single chain diabodies, bispecific T cell engagers and various other derivatives of these (for example Byrne H. et al., 2013, Trends Biotech, 31(11):621-632, showing various bispecific antibody formats). Several bispecific antibody formats can redirect effector cells to target cells that play a key role in the disease process. For example, several bispecific antibody formats can retarget effector cells to B cells, and a variety of bispecific antibody constructs are designed to retarget cells of the immune system, for example by binding to and triggering Fc receptors on the surface of effector cells or by binding to T cell receptor complexes. Figure 2 Several bispecific antibody formats can redirect effector cells to target cells that play a key role in the disease process. For example, several bispecific antibody formats can retarget effector cells to B cells, and a variety of bispecific antibody constructs are designed to retarget cells of the immune system, for example by binding to and triggering Fc receptors on the surface of effector cells or by binding to T cell receptor complexes.

[0098] The antibody used according to the present application can be in any antibody format. Examples of bispecific antibody formats include, but are not limited to, tetrabodies, chemically conjugated Fabs (fragment antigen binding) and BiTE® (bispecific T cell engager). In one embodiment of the present application, the antibody used is preferably a BiTE® (bispecific T cell engager) BiTE® (bispecific T cell engager).

[0099] Thus, the antibody used according to the present application can be selected from the group consisting of Triomabs; hybrid hybridomas (tetrabodies); multispecific anticalin platform (Pieris); diabodies; single-chain diabodies; tandem single-chain Fv fragments; TandAbs, tri-specific Abs (Affimed) (105-110 kDa); Darts (dual affinity re-targeting; Macrogenics); bispecific Xmabs (Xencor); bispecific T cell engagers (Bites; Amgen; 55 kDa); Triomabs; Triomabs = Fab-scFv fusion proteins (Creative Biolabs) multifunctional recombinant antibody derivatives (110 kDa); Duobody platform (Genmab); Dock and lock platform; Knob into hole (KIH) platform; humanized bispecific IgG antibodies (REGN1979) (Regeneron); Mab2 bispecific antibodies (F-Star); DVD-Ig = dual variable domain immunoglobulin (Abbvie); kappa-lambda bodies; TBTI = tetravalent bispecific tandem Ig; and CrossMab.

[0100] The antibody used according to the present application can be selected from the group consisting of bispecific IgG-like antibodies, including CrossMab; DAF (two-in-one); DAF (four-in-one); DutaMab; DT-IgG; common knob-into-hole LC; knob-into-hole assembly; Charge pair; Fab arm exchange; SEEDbody; Triomab; LUZ-Y; Fcab; and orthogonal Fab. These bispecific antibody formats are shown and described in Spiess C., Zhai Q. and Carter P.J. (2015) Molecular Immunology 67:95-106, in particular Figure 1 and the corresponding text description (e.g. p. 95-101).

[0101] The antibody used according to the present application can be selected from the group consisting of IgG appended antibodies with additional antigen binding moieties, comprising DVD-IgG; IgG(H)-scFv; scFv-(H)IgG; IgG(L)-scFv; scFV-(L)IgG; IgG(L,H)-Fv; IgG(H)-V; V(H)-IgG; IgG(L)-V; V(L)-IgG; KIH IgG-scFab; 2scFv-IgG; IgG-2scFv; scFv4-Ig; scFv4-Ig; Zybody; and DVI-IgG (Tetrabody). These bispecific antibody formats are shown and described in Spiess C., Zhai Q. and Carter P.J. (2015) Molecular Immunology 67: 95-106, in particular Figure 1 and the corresponding text description (e.g. p. 95-101).

[0102] The antibody used according to the present application can be selected from the group consisting of bispecific antibody fragments, comprising Nanobody; Nanobody-HAS; BiTE; Diabody; DART; TandAb; scDiabody; sc-Diabody-CH3; Diabody-CH3; Triabody; Miniantibody; Minibody; TriBi minibody; scFv-CH3 KIH; Fab-scFv; scFv-CH-CL-scFv; F(ab’)2; F(ab’)2-scFv2; scFv-KIH; Fab-scFv-Fc; Tetravalent HCAb; scDiabody-Fc; Diabody-Fc; Tandem scFv-Fc; and Intrabody. These bispecific antibody formats are shown and described in Spiess C., Zhai Q. and Carter P.J. (2015) Molecular Immunology 67: 95-106, in particular Figure 1 and the corresponding text description (e.g. p. 95-101).

[0103] The antibody used according to the present application can be selected from the group consisting of bispecific fusion proteins, comprising Dock and lock; ImmTAC; HSAbody; scDiabody-HAS; and Tandem scFv-Toxin. These bispecific antibody formats are shown and described in Spiess C., Zhai Q. and Carter P.J. (2015) Molecular Immunology 67: 95-106, in particular Figure 1and shown and described in the corresponding written description (e.g. p. 95-101).

[0104] The antibody used according to the present application can be selected from the group consisting of bispecific antibody conjugates comprising IgG-IgG; Cov-X-Body; and scFvl-PEG-scFv2. These bispecific antibody formats are described in e.g. Spiess C., Zhai Q. and Carter P.J. (2015) Molecular Immunology 67: 95-106, in particular Figure 1 and shown and described in the corresponding written description (e.g. p. 95-101).

[0105] It is also preferred that the antibody used according to the present application is selected from the group consisting of bispecific T cell engagers (BiTE TM ) and bispecific tri-functional antibodies.

[0106] It is also preferred that the antibody used according to the present application comprises at least two different single chain variable fragments (scFv). An scFv is herein to be understood as a fusion protein of the variable regions of the heavy chain (VH) and the light chain (VL) of an immunoglobulin connected with a short linker peptide. The peptide usually comprises at least 5, preferably at least 10, more preferably about 25 amino acids. The linker is usually rich in glycine for flexibility, and in serine or threonine for solubility, and can connect the N-terminus of the VH with the C-terminus of the VL, or vice versa. Despite the removal of the constant regions and the introduction of the linker, the scFv can retain the specificity of the original immunoglobulin. Usually, scFv can be produced directly from subcloned heavy and light chains derived from a hybridoma. ScFv are often used in e.g. flow cytometry, immunohistochemistry, and as antigen binding domains of artificial T cell receptors. In the context of the present application, they are preferably used as antigen binding domains of artificial T cell receptors.

[0107] It is preferred that the antibody used according to the present application has an IgG-like format (based on IgG, also referred to as “IgG-type”), wherein an antibody having an IgG-like format usually comprises two heavy chains and two light chains. Generally, immunoglobulin G (IgG) is a type of antibody. It is herein to be understood as a protein complex consisting of four peptide chains arranged in a Y-shape typical for antibody monomers of two identical heavy chains and two identical light chains. Each IgG usually has two antigen binding sites, which can be different or identical. Representing about 75% of serum antibodies in humans, IgG is the most common type of antibody found in circulation. Physiologically, IgG molecules are produced and released by plasma B cells.

[0108] Examples of antibodies with IgG-like formats include hybridomas and various IgG-scFv formats (see: Byrne H. et al. (2013) Trends Biotech, 31(11):621-632; Figure 2 A-E), wherein tetrameric tumors are preferred, which are preferably produced by fusion of two different hybridomas. In the IgG class, the antibody can be preferably based on the IgG1, IgG2, IgG3 or IgG4 subclass, wherein antibodies based on IgG1 are preferred (also referred to as "IgG1 type"). The multispecific antibodies or antigen binding fragments used according to the present application, e.g. bispecific antibodies, can also be based on any immunoglobulin class (e.g. IgA, IgG, IgM, etc.) and subclass (e.g. IgA1, IgA2, IgG1, IgG2, IgG3, IgG4, etc.).

[0109] Preferred bispecific IgG-like antibody formats include, for example, hybrid hybridomas (tetrameric tumors), common light chain architecture, various IgG-scFv formats, various scFv-IgG formats, two-in-one IgG, dual V domain IgG, IgG-V and V-IgG, e.g. in Chan, A.C. and Carter, P.J. (2010) Nat Rev Immu 10:301-316. Figure 3 c and described in said article. Other preferred bispecific IgG-like antibody formats include, for example, DAF, CrossMab, IgG-dsscFv, DVD, IgG-dsFV, IgG-scFab, scFab-dsscFv and Fv2-Fc, which are described in Spiess C., Zhai Q. and Carter P.J. (2015) Molecular Immunology 67:95-106. Figure 1Further preferred bispecific IgG-like antibody formats include DAF (two-in-one); DAF (four-in-one); DutaMab; DT-IgG; tetrabody; charged pair; Fab arm exchange; SEEDbody; Triomab; LUZ-Y; Fcab; orthogonal Fab; DVD-IgG; IgG(H)-scFv; scFv-(H)IgG; IgG(L)-scFv; scFV-(L)IgG; IgG(L,H)-Fv; IgG(H)-V; V(H)-IgG; IgG(L)-V; V(L)-IgG; KIH IgG-scFab; 2scFv-IgG; IgG-2scFv; scFv4-Ig; scFv4-Ig; Zybody; and DVI-IgG (four-in-one), e.g. in Spiess C., Zhai Q. and Carter P.J. (2015) Molecular Immunology 67:95-106, in particular Figure 1 and the corresponding text description (e.g. p. 95-101) shown and described.

[0110] Trifunctional bispecific antibodies for use according to the present application can be produced by three main methods: (i) chemical conjugation, which involves chemical cross-linking; (ii) fusion of two different hybridoma cell lines; or (iii) genetic methods involving recombinant DNA technology. Fusion of two different hybridomas produces hybrid-hybridomas (or "tetrabodies") that secrete a heterogeneous population of antibodies comprising bispecific molecules.

[0111] Other alternative methods can include chemical conjugation of two different mAbs and / or smaller antibody fragments. Oxidative recombination strategies to link two different antibodies or antibody fragments have been found to be inefficient due to side reactions during re-oxidation of multiple native disulfide bonds. Current chemical conjugation methods focus on the use of homobifunctional or heterobifunctional cross-linkers. Recombinant DNA technology produces the widest range of bispecific antibodies by artificial manipulation of genes and represents the most diverse method of bispecific antibody generation (45 formats in the last two decades; see Byrne H. et al. (2013) Trends Biotech, 31(11):621-632).

[0112] Regimen of the method of treatment

[0113] In the context of the trifunctional bispecific antibody for use in the application, a patient with an EBV infection will be identified. Due to the reactivation of EBV in B cells, the patient can suffer from one or more autoimmune diseases selected from the group consisting of rheumatoid arthritis, Hashimoto's disease, type 1 diabetes and multiple sclerosis, or one or more chronic inflammatory diseases selected from the group consisting of chronic prostatitis, chronic cystitis, chronic hepatitis (non-alcoholic), irritable bowel syndrome (IBS), chronic neuroinflammation and metabolic syndrome, or one or more diseases and conditions selected from the group consisting of Sjogren's syndrome, myasthenia gravis, Crohn's disease, vitiligo, type 2 diabetes, chronic inflammation of the milk duct, lichen in the vagina, chronic pancreatitis, chronic bronchitis and chronic flue-like symptoms, chronic fatigue syndrome, chronic dry cough, chronic rhinitis, night sweats, sleep disorders; insomnia, multiple neuropathic pain, edema (e.g. in fingers, toes and face), endometriosis, ovarian cysts, irregular menstruation, hair loss, memory problems, dry eyes and dry mouth, migraine, common hair loss, acne, weight gain, poor concentration, insulin resistance, diarrhea and chronic herpes zoster.

[0114] The disease and / or condition can also be one or more of depression, allergic rhinitis, chronic asthma, lactose and gluten allergy, multiple allergies (mainly runny nose, itching), psoriasis, bladder dysfunction and secondary open-angle glaucoma.

[0115] In a first step, autologous B cells are isolated from a patient suffering from EBV reactivation. For example, these autologous B cells can be obtained from autologous mononuclear cells (PBMC) of peripheral blood isolated from the patient, and then the autologous B cells are enriched from the isolated PBMC.

[0116] In a second step, the enriched B cells are incubated with the trifunctional bispecific antibody of the application for a period of time sufficient to establish a physical interaction between the trifunctional bispecific antibody and the enriched B cells, to obtain an incubation mixture, wherein the trifunctional bispecific antibody comprises: (a) a first binding arm binding to a B cell via a B cell surface antigen; (b) a second binding arm binding to a T cell via a T cell surface antigen; (c) a Fc part binding to a Fc receptor positive cell. The incubation time is preferably between 1 min and 60 min. More preferred incubation times have been disclosed above, wherein an incubation time of 10 min to 15 min will result in an excellent binding affinity between the antibody and the B cells.

[0117] In a third step, the incubation mixture obtained from the second step is transferred back into the same patient from which the B cells or PBMCs were provided. PBMCs are any peripheral blood cells and comprise B cells, T cells, natural killer cells and monocytes. These cells can be isolated from whole blood by conventional methods known to the skilled person, for example by performing standard Ficoll density centrifugation. After isolation of the PBMCs, the B cells comprised in the PBMCs can be enriched by conventional methods known to the person skilled in the art, for example by using appropriate immunomagnetic beads. The PBMCs and B cells can be kept in conventional cell culture medium for incubation, wherein the physical binding between the antibody and the B cells or PBMCs starts. Subsequently, chromogenic in situ hybridization can be performed to detect the expression of EBV-specific RNA in the enriched B cells to identify the infection of EBV in the B cells. Preferably, the EBV-specific RNA is, but is not limited to, EBER, EBNA1 or EBNA2 RNA.

[0118] Preferably, the above-mentioned second step further comprises an autologous cell preparation of mononuclear cells of PBMCs of the same patient, wherein the PBMCs are added to the incubation mixture of the enriched B cells and the trifunctional bispecific antibody for a time period sufficient to establish a physical interaction between the PBMCs and the trifunctional bispecific antibody, wherein the time period is preferably between 1 min and 60 min. More preferred incubation times have been disclosed above, wherein an incubation time of 10 min to 15 min will result in an excellent binding affinity between the antibody and the B cells.

[0119] The PBMCs and the autologous B cells can be mixed and incubated with the antibody simultaneously or consecutively.

[0120] In the above-mentioned third step, the trifunctional bispecific antibody bound to the enriched B cells, preferably also to the PBMCs, is transferred back into the patient from which the B cells or PBMCs were provided.

[0121] Upon application back into the patient, for example subcutaneously, the antibody will initiate the activation of the bound immune cells via binding to CD3 on, for example, T cells and Fc gamma receptors on, for example, accessory immune cells. These interactions will result in the phagocytosis of the viral material of the involved EBV-infected B cells and in the processing of the viral material within Fc receptor-positive, professional antigen-presenting cells. Eventually, the virus-specific peptides will be presented to T cells with the appropriate T cell receptors via MHC class I and II molecules. All these interactions eventually lead to a polyclonal humoral and cellular anti-EBV immune response. This reaction is beneficial for the patient to better control the EBV infection and to prevent the patient from suffering from diseases associated with the reactivation of EBV.

[0122] For use of the trifunctional bispecific antibody in the context of the present application, the antibody can be administered during incubation preferably in an amount of 0.1-100 pg, more preferably in an amount of 0.4-50 pg, further preferably in an amount of 0.6-20 pg, even further preferably in an amount of 0.8-10 pg. This is also the amount administered to the patient.

[0123] In a second aspect of the present application, a pharmaceutical composition comprising an antibody for use in a method for treating a patient suffering from a disease and / or disorder associated with reactivation of EBV in B cells according to the first aspect of the present application is disclosed, the method comprising: providing an autologous cell preparation of enriched B cells of the patient; incubating the enriched B cells with a trifunctional bispecific antibody for a period of time sufficient to establish a physical interaction between the trifunctional bispecific antibody and the enriched B cells to obtain an incubation mixture; transferring the incubation mixture obtained after incubation into the same patient, wherein the trifunctional bispecific antibody comprises: (a) a first binding arm binding to a B cell via a B cell surface antigen; (b) a second binding arm binding to a T cell via a T cell surface antigen; (c) a Fc part binding to an Fc receptor positive cell, and wherein the disease and / or disorder associated with reactivation of EBV in B cells is selected from one or more autoimmune diseases of rheumatoid arthritis, Hashimoto's disease, type 1 diabetes mellitus and multiple sclerosis, or wherein the disease and / or disorder associated with reactivation of EBV in B cells is selected from one or more chronic inflammatory diseases of chronic prostatitis, chronic cystitis, chronic hepatitis (non-alcoholic), irritable bowel syndrome (IBS), chronic neuroinflammation and metabolic syndrome, or wherein the disease and / or disorder associated with reactivation of EBV in B cells is selected from one or more diseases and disorders of Sjogren's syndrome, myasthenia gravis, Crohn's disease, vitiligo, type 2 diabetes mellitus, chronic inflammation of the milk duct, lichen in the vagina, chronic pancreatitis, chronic bronchitis and chronic flue-like symptoms, chronic fatigue syndrome, chronic dry cough, chronic rhinitis, night sweats, sleep disorders; insomnia, multiple neuropathic pain, edema (e.g. in fingers, toes and face), endometriosis, ovarian cysts, irregular menstruation, hair loss, memory problems, dry eyes and dry mouth, migraine, common hair loss, acne, weight gain, poor concentration, insulin resistance, diarrhea and chronic shingles

[0124] Preferably, the pharmaceutical composition used further comprises a pharmaceutically acceptable carrier and / or excipient, including for example binding agents, lubricants, disintegrants, fillers and diluents.

[0125] The pharmaceutical composition used comprises an autologous cell preparation of enriched B cells of a patient having a disease and / or disorder associated with reactivation of EBV in B cells according to the first aspect of the application. The enriched B cells can be prepared by any of the methods described above.

[0126] In another embodiment of the application, the pharmaceutical composition used according to the application further comprises an autologous cell preparation of PBMCs of the same patient, wherein the PBMCs are added to the incubation mixture of the enriched B cells and the trifunctional bispecific antibody for a period of time sufficient to establish a physical interaction between the PBMCs and the trifunctional bispecific antibody prior to transferring the incubation mixture into the same patient. As described above, the PBMCs can be isolated from whole blood, e.g. by performing standard Ficoll density centrifugation.

[0127] The PBMCs and the autologous B cells can be mixed and incubated with the antibody simultaneously or consecutively.

[0128] The period of time for incubation of the B cells and the antibody, the period of time for incubation of the PBMCs and the antibody and the amount of antibody used for incubation can be applied as described before.

[0129] In yet another embodiment, the pharmaceutical composition used according to the application is in the form of a kit-of-parts comprising the trifunctional bispecific antibody as disclosed above, a preparation of enriched autologous B cells from a patient infected with EBV, preferably a preparation of autologous PBMCs from the same patient, wherein at least two components are provided in separate containers. The B cells are incubated with the trifunctional bispecific antibody for a period of time sufficient to establish a physical interaction between the trifunctional bispecific antibody and B cells, thereby obtaining an incubation mixture. Preferably, the PBMCs are added to the incubation mixture of the enriched B and the trifunctional bispecific antibody for a period of time sufficient to establish a physical interaction between the PBMCs and the trifunctional bispecific antibody prior to transferring the incubation mixture into the same patient. The period of time for incubation of the B cells and the antibody, the period of time for incubation of the PBMCs and the antibody and the amount of antibody used for incubation can be applied as described before. The PBMCs and the autologous B cells can be mixed and incubated with the antibody simultaneously or consecutively.

[0130] In a third aspect of the application, an ex vivo method for the preparation of a pharmaceutical composition for use in a method of treating a patient having a disease and / or disorder associated with reactivation of EBV in B cells according to the second aspect of the application is disclosed, the method comprising: providing an autologous cell preparation of enriched B cells of the patient; incubating the enriched B cells with a trifunctional bispecific antibody for a period of time sufficient to establish a physical interaction between the trifunctional bispecific antibody and the enriched B cells to obtain an incubation mixture, wherein the trifunctional bispecific antibody comprises: (a) a first binding arm binding to a B cell via a B cell surface antigen; (b) a second binding arm binding to a T cell via a T cell surface antigen; (c) a Fc part binding to Fc receptor positive cells, and wherein the disease and / or disorder associated with reactivation of EBV in B cells is selected from one or more autoimmune diseases consisting of rheumatoid arthritis, Hashimoto's disease, type 1 diabetes and multiple sclerosis, or wherein the disease and / or disorder associated with reactivation of EBV in B cells is selected from one or more autoimmune diseases consisting of chronic prostatitis, chronic cystitis, chronic hepatitis (non-alcoholic), irritable bowel syndrome (IBS), chronic neuroinflammation and metabolic syndrome; or wherein the disease and / or disorder associated with reactivation of EBV in B cells is selected from one or more diseases and disorders of Sjogren's syndrome, myasthenia gravis, Crohn's disease, vitiligo, type 2 diabetes, chronic inflammation of the milk duct, lichen of the vagina, chronic pancreatitis, chronic bronchitis and chronic flue-like symptoms, chronic fatigue syndrome, chronic dry cough, chronic rhinitis, night sweats, sleep disorders; insomnia, multiple neuropathic pain, edema, e.g. of the fingers, toes and face, endometriosis, ovarian cysts, irregular menstruation, hair loss, memory problems, dry eyes and dry mouth, migraine, common hair loss, acne, weight gain, poor concentration, insulin resistance, diarrhea and chronic shingles. Preferably, the B cells are prepared by any of the methods described above. The period of time the autologous B cells are incubated with the trifunctional bispecific antibody can be from 1 min to 60 min, preferably from 1 min to 20 min, more preferably from 5 min to 20 min, even more preferably from 8 min to 15 min, further preferably from 10 min to 15 min, e.g. 10, 11, 12, 13, 14 or 15 min. Preferably, the antibody is used in an amount as disclosed above.

[0131] Preferably, the ex vivo method further comprises adding PBMCs of the same patient having EBV reactivation in B cells to the above-mentioned incubation mixture of the enriched autologous B cells and the trifunctional bispecific antibody for a period of time sufficient to establish a physical interaction between the trifunctional bispecific antibody and the PBMCs. The PBMCs and the autologous B cells can be mixed and incubated with the antibody simultaneously or consecutively. The PBMCs can be isolated from whole blood by, for example, performing standard Ficoll density centrifugation. The period of time during which the PBMCs are incubated with the incubated autologous B cells and the trifunctional bispecific antibody can be 1 min to 60 min, preferably 1 min to 20 min, more preferably 5 min to 20 min, even more preferably 8 min to 15 min, further preferably 10 min to 15 min, e.g. 10, 11, 12, 13, 14 or 15 min.

[0132] In the present application, the trifunctional bispecific antibody is capable of recruiting and activating (i) T cells and (ii) Fc receptor expressing cells, such as accessory immune cells, e.g. monocytes, macrophages, natural killer cells, dendritic cells and / or activated neutrophils and other Fc receptor expressing cells, and (iii) targeted B cells. The simultaneous activation of these different types of effector cells leads to an efficient killing of EBV infected B cells by various mechanisms, such as phagocytosis and perforin-mediated cytotoxicity. In general, the net effect of a trifunctional antibody comprising Fc receptors is to link T cells and Fc receptor positive cells to the target B cells, i.e. EBV infected B cells, leading to the destruction of the virus-infected cells.

[0133] The trifunctional antibody causes the removal of targeted cells in particular by (i) antibody-dependent cell-mediated cytotoxicity, (ii) T cell-mediated cell killing and (iii) induction of anti-viral immunity. However, only the first mode of action is actually performed by conventional (monoclonal and monospecific) antibodies. In contrast to conventional antibodies, the trifunctional bispecific antibodies in the present application have a higher cytotoxic potential and they even bind to relatively weakly expressed antigens. Thus, the trifunctional bispecific antibodies in the present application are more effective (more than 1,000-fold) in eliminating targeted cells at an equivalent dose compared to conventional antibodies.

[0134] The above-mentioned embodiments can be combined in any way, if appropriate. Further possible embodiments and implementation forms of the present application also include combinations of features which are not explicitly mentioned in the foregoing or in the following description of the examples of the present application. In particular, a person skilled in the art will also add individual aspects as improvements or supplements to the respective basic form of the present application.

[0135] In the following, individual examples of the present application will be described in detail. These examples are illustrative and do not limit the scope of the present application.

[0136] Materials and methods:

[0137] PBMC preparation and B cell enrichment

[0138] Mononuclear cells (PBMC) from peripheral blood were isolated from non-coagulated EDTA or heparin blood by standard Ficoll density centrifugation using human Pancoll solution (Pan Biotech, Germany). Isolated PBMC were washed twice with phosphate buffered saline (PBS) (Pan Biotech, Germany). Red blood cells were lysed using red blood cell lysis buffer. Approximately 1 x 10Exp6PBMC can be isolated from 1 ml blood. B cells of the PBMC preparation were enriched by immunomagnetic beads using the Pan Mouse IgG kit (Dynal Biotech, Germany): First, B cells of the PBMC preparation (1 x 10Exp7 / ml) were labeled with an anti-CD20 monoclonal mouse antibody (TPA10, Trion Research). Then, the labeled B cells were isolated by adding magnetic beads coupled to anti-mouse IgG. Since the capture antibody is linked to the beads via a DNA linker, the captured B cells can be released from the beads by enzymatic cleavage with DNAse. Finally, the cells were washed several times with PBS until no residual beads were visible. The efficiency of B cell enrichment was controlled by FACS analysis and usually ranged between 70-99%. TM Pan Mouse IgG kit (Dynal Biotech, Germany): First, B cells of the PBMC preparation (1 x 10Exp7 / ml) were labeled with an anti-CD20 monoclonal mouse antibody (TPA10, Trion Research). Then, the labeled B cells were isolated by adding magnetic beads coupled to anti-mouse IgG. Since the capture antibody is linked to the beads via a DNA linker, the captured B cells can be released from the beads by enzymatic cleavage with DNAse. Finally, the cells were washed several times with PBS until no residual beads were visible. The efficiency of B cell enrichment was controlled by FACS analysis and usually ranged between 70-99%.

[0139] Colorimetric in situ hybridization (CISH) for detection of EBV-specific EBER RNA

[0140] Potential EBV-infected B cells and PBMC expressing untranslated EBV-specific EBER1 and EBER 2 RNA were detected by CISH using digoxigenin-labeled EBER RNA-specific oligonucleotide probes (Zytovision, ZytoFast EBV probes, IVD Medical Devices, Germany). PBMC and B cells were prepared as described above. In situ hybridization was performed according to the manufacturer's instructions. Cytospins were analyzed by microscopy. Positively stained cells appeared red.

[0141] Detection of gp350 antigen on B cells

[0142] Cytospin slides were analysed by counting the positively red stained cells by a computer image analysis system (MDS, Applied Imaging).

[0143] Cytokine quantification

[0144] Plasma samples were analysed for cytokine levels using the Luminex system 200 (Luminex, TX, USA) and pre-mixed 8-plex fluorokine X-Map kits (R&D Systems, MN, USA) containing the cytokines IL-2, IL-4, IL-6, IL-8, IL-10, IL-17, IFN-gamma and TNF-alpha. Samples were collected at the indicated time points, stored at -20°C and measured in batches. The limit of detection for cytokines was 3.2 pg / ml.

[0145] Detection of anti-EBNA-1 and VCA IgG antibodies

[0146] Antibodies in patient plasma specific for the EBV antigens EBNA-1 (Epstein-Barr nuclear antigen 1) and VCA (viral capsid antigen) were determined by the IVD kit ELISA according to the manufacturer's (medac GmbH, Germany) instructions. Briefly, plasma samples were incubated on pre-coated and pre-blocked microtiter plates. After a washing step, bound EBV-specific IgG antibodies were detected by peroxidase-coupled anti-human IgG antibodies. Finally, the washed plates were developed using a TMB (tetramethylbenzidine) substrate solution and then the reaction was stopped with sulfuric acid. The microtiter plates were measured at 450 nm using a Versamax plate reader (Molecular Devices, USA). EBV-specific antibody concentrations were calculated by interpolation on a standard curve. Results of > 11 AU / ml were considered positive.

[0147] Preparation of autologous cell vaccines

[0148] PBMC and B cell enrichment from 60 ml EDTA peripheral blood was performed as described above. All manipulations were performed under sterile conditions under laminar air flow. 250 x 10 3 Cells of the B cell fraction (in 0.5 ml PBS) and 500 x 10 3PBMC (in 0.5 ml PBS) were loaded into separate sterile gasket-sealed glass vials. Then, 1 μg of the trifunctional bispecific antibody with anti-CD20 x anti-CD3 specificity (in 0.1 ml PBS) was added to the B cell fraction and the cells were incubated for 10 min at room temperature. Subsequently, the PBMC fraction was added to the pre-incubated autologous B cell fraction and incubated for another 10 min. Finally, the whole cell preparation was applied subcutaneously back to the patient. For boost applications, the same procedure was repeated. Example

[0149] The application will now be described in detail with reference to examples thereof. These examples are illustrative and do not limit the scope of the application.

[0150] Example 1. Trifunctional bispecific antibody mediated in vitro killing of enriched autologous B cells

[0151] Example 1 demonstrates the efficient killing of enriched autologous B cells targeted in vitro by a CD20 specific trifunctional bispecific antibody. As described in the methods and materials section, 250.000 enriched B cells and 500.000 PBMC from a healthy donor were prepared. The cells were mixed and incubated in the presence or absence of 1 μg Bi20 as trifunctional bispecific anti-CD3 x anti-CD20 antibody (see Figure 1 ). The cells were incubated in a total volume of 1 ml medium (RPMI 1640 medium supplemented with 8.9% FCS, 2 mM L-glutamine, 1 mM sodium pyruvate and 1 x non-essential amino acids) in 24 well plates at 37°C and 5% CO2. After three days, the cells were analyzed by flow cytometry using a FACS-Calibur and Cellquest pro software (Becton Dickinson, USA). B cells were stained by a PE (phycoerythrin) conjugated anti-human CD20 monoclonal antibody (Caltag, USA) and quantified by histogram statistics. As shown in Figure 2 Example 1, the B cell enrichment efficiency was more than 80%. After three days, the B cell population detected in the procedure without Bi20 averaged 9.24% (n=2; Figure 2 B). In contrast, in the procedure where Bi20 was added, only an average of 3.75% B cells (n=2) were quantified Figure 2 C). Indeed, comparison of the histograms demonstrated that the different CD20+ B cell population was completely eliminated. Thus, the addition of the trifunctional bispecific antibody Bi20 efficiently and completely eliminated the targeted B cells by retargeted cytotoxicity.

[0152] Example 2 (chronic fatigue syndrome, chronic dry cough, night sweats)

[0153] A 49-year-old woman with EBV test positive suffered from episodes of fatigue for ten years, lymphedema, chronic otitis media, frequent night sweats. In addition, the patient was diagnosed with pancreatitis in May 2010 and developed rectal cancer in July 2010. EBV was detected in tumor cells by PCR analysis and subsequently in the surgical scar. Since 2000, the patient has suffered from chronic dry cough.

[0154] In a detailed EBV diagnosis, a high frequency of EBER-1+2 CISH positive B cells (>50%) as well as a slightly elevated IL-8 value could be determined, for example, in the patient's enriched B cell fraction.

[0155] Due to this diagnosis and unmet medical need, the patient decided to participate in a compassionate use treatment with a research-based anti-EBV therapeutic vaccine.

[0156] To prepare the vaccine cell preparation, 60 ml EDTA peripheral blood was taken from the patient. First, the lymphocytes were separated by Ficoll gradient centrifugation. Subsequently, the B cells were enriched from the fraction of isolated PBMCs using CD20-specific antibodies by immunomagnetic beads.

[0157] After DNase digestion and several washing steps to remove the immunomagnetic beads, an enriched B cell fraction (>85%) can be produced.

[0158] Then 250 x 10 3 B cell fraction cells (in 0.5 ml sterile PBS) were incubated with 1 μg (0.1 ml PBS) Bi20 (trifunctional bispecific antibody with anti-CD20 x anti-CD3 specificity) for 10 minutes.

[0159] Subsequently, 500 x 10 3 PBMCs (in 0.5 ml sterile PBS) were added to the pre-incubated autologous B cell fraction and incubated for another 10 minutes.

[0160] All manipulations were carried out under sterile conditions at room temperature under laminar air flow.

[0161] Finally, the entire cell preparation was applied subcutaneously back to the patient (15 years, February 26th).

[0162] For the booster application, the same steps were repeated (15 years, March 25th and 16 years, February 2nd).

[0163] The following diagnostic parameters were evaluated before, during and after the treatment:

[0164] Immunological results:

[0165] Cytokine measurements

[0166]

[0167]

[0168] Humoral anti-EBV response

[0169]

[0170] PCR analysis of EBV

[0171]

[0172] * Analysis with PBMCs of the patient isolated by Ficoll gradient

[0173] Clinical results:

[0174] After the second application of the cell preparation (booster), the cough improved and the EBV was detected negative in the surgical scar and in the blood (PCR analysis). In addition, the patient described an improvement of night sweats and felt more energetic and an improvement of the concentration ability. In addition, since the last treatment, no severe infections were observed and a normalization of the bowel movements was observed. The EBV infected B cells in the patient were significantly reduced Figure 3

[0175] Example 3 (insulin resistance, type 2 diabetes, potency problems)

[0176] A 50 year old male, positive for EBV and 1, 2 herpes virus, CMV and chlamydia pneumonia, suffers from a metabolic syndrome with high insulin resistance, elevated liver enzymes, abnormal liver metabolism, hypercholesterolemia, high triglycerides, sleep disorders, fatigue, poor concentration and potency problems.

[0177] In a detailed EBV diagnosis, a high frequency of EBNA-1+2 CISH positive B cells (50-75%) can be determined, for example in the enriched B cell fraction of the patient. Due to this diagnosis and the unmet medical need, the patient decided to participate in a compassionate treatment with the investigational anti-EBV therapeutic vaccine, also as described in example 2.

[0178] A cell preparation similar to examples 1 and 2 was generated ex vivo and applied subcutaneously back to the patient (May 13, 2015).

[0179] For the booster application, the same steps were repeated (June 10, 2015).

[0180] HOMA (<2) February 24, 2015 6.6 May 13, 2015 6.0 July 29, 2015 4.5

[0181] ​HOMA: Homeostasis Model Assessment, HOMA index = insulin (fasting, μU / ml) x glucose (fasting, mg / dl) / 405, HOMA index > 2 is an indicator for insulin resistance.

[0182] Clinical results:

[0183] The patient shows improved fatigue and performance symptoms, liver enzymes, triglycerides and improved cholesterol values and insulin resistance. The patient can sleep better. EBV infected B cells in the patient are significantly reduced Figure 4

[0184] Example 4 (skin inflammation, sleep disorders, repeated infections)

[0185] A 41 year old female with chronic bronchitis, metabolic syndrome, dystonia vegetativa, extreme fatigue, constipation and large amounts of gas, lactose intolerance, weight loss, sleep disorders, chronic flue, large scale acne-like skin problems on the face and back, night sweats, submandibular and cervical lymph node enlargement suffers from a positive EBV (PCR) test. In addition, the test for herpes-1 is positive with PCR. IgG titers for CMV, herpes-6 and herpes-1 and -2 are high.

[0186] In a detailed EBV diagnosis, e.g. in the patient's enriched B cell fraction, a high frequency of EBNA-1+2 CISH positive B cells (> 75%) as well as slightly elevated IL-8 values can be determined.

[0187] Due to this diagnosis and unmet medical need, the patient decides to participate in a compassionate use treatment with a research anti-EBV therapeutic vaccine as described in example 2.

[0188] A cell preparation similar to examples 1 and 2 is generated ex vivo and applied subcutaneously back to the patient (15 March 26).

[0189] For the booster application the same procedure is repeated (15 May 5).

[0190] The following diagnostic parameters are assessed before, during and after the treatment:

[0191] Immunological results:

[0192] Cytokine measurements

[0193]

[0194] Humoral anti-EBV response

[0195] ​

[0196] Hematological results:

[0197] PCR analysis of EBV

[0198] Date January 14, 2015 August 31, 2015 EBV-specific PCR Positive (30.58) 33.71

[0199] Analysis with patient's PBMC isolated by Ficoll gradient

[0200] Enriched B cells (CD20, 2.5 x 10e5) were stained with anti-gp350 antibody APAAP technique

[0201]

[0202] Clinical results:

[0203] Improvement of skin inflammation and fatigue symptoms were observed. Bronchial infection rate was significantly reduced. The patient could sleep normally again without night sweats. Body weight as well as vegetative dystonia improved (neurogastro improved) with normalization of defecation. EBV infected B cells in the patient were significantly reduced Figure 5

[0204] Example 5 (Multiple neuropathic pain, swelling of fingers, toes and face, repeated infections, sleep disorders, lymph node enlargement, endometriosis, chronic cystitis, chronic inflammation of the milk ducts)

[0205] A 48 year old female, positive for EBV test, suffered from endometriosis, fatigue, memory and concentration problems, sleep problems, night sweats, inguinal and submandibular lymph node enlargement, chronic cystitis, chronic inflammation of the milk ducts in both breasts, multiple neuropathic pain in fingers and toes, lymphedema of the legs, fingers and face and allergies (runny nose). The patient developed bladder cancer (stage 1) in 2014. The patient showed high IGG for herpes type 6, CMV and chlamydia pneumonia.

[0206] In a detailed EBV diagnosis, for example in the enriched B cell fraction of the patient, a high frequency of EBER-1+2 CISH positive B cells (> 75%) as well as slightly elevated IL-8 values could be determined.

[0207] Due to this diagnosis and unmet medical need, the patient decided to participate in a compassionate use treatment with a research anti-EBV therapeutic vaccine as described in example 2.

[0208] Cellular preparations similar to examples 1 and 2 were generated ex vivo and applied subcutaneously back to the patient (March 24, 2015).

[0209] For the booster application the same steps were repeated (May 5, 2015). ​

[0210] The following diagnostic parameters are evaluated before, during and after treatment:

[0211] Immunological results:

[0212] Cytokine measurements

[0213]

[0214] Humoral anti-EBV response

[0215]

[0216] Enriched B-cells (CD20, 2.5 x 10e5) stained with anti-gp350 antibody APAAP technique

[0217]

[0218] Clinical results:

[0219] After the boost, the patient shows an improvement in concentration and memory as well as normalization of sleep. Chronic inflammation in the milk ducts of the breast as well as fatigue symptoms disappear. Multiple neuropathic pain as well as swelling of the fingers, toes and face improve. Also an improvement in allergic symptoms (runny nose) and a decrease in infection rate are observed. EBV infected B-cells in the patient significantly decrease Figure 6 .

[0220] Example 6 (insulin resistance, type 2 diabetes, Sjogren's syndrome, rheumatoid arthritis, Hashimoto's disease, hair loss, memory problems, dry eyes and mouth)

[0221] A 58 year old female, positive for EBV test, suffers from Sjogren's syndrome, rheumatoid arthritis, Hashimoto's disease, hair loss, memory problems, dry eyes and mouth, insulin resistance.

[0222] In a detailed EBV diagnosis, a high frequency of EBER-1+2 CISH positive B-cells (50-75%) can be determined, for example in the enriched B-cell fraction of the patient.

[0223] Due to this diagnosis and unmet medical need, the patient decides to participate in a compassionate use treatment with the investigational anti-EBV therapeutic vaccine as described in example 2.

[0224] A cell preparation similar to examples 1 and 2 is generated ex vivo and applied subcutaneously back to the patient (15 July 29).

[0225] For the boost application, the same procedure is repeated (15 September 15).

[0226] The following diagnostic parameters are evaluated before, during and after treatment:

[0227] Immunological results:

[0228] Cytokine measurements

[0229]

[0230] Humoral anti-EBV response

[0231]

[0232] Enriched B cells (CD20, 2.5 x 10e5) stained with anti-gp350 antibody APAAP technique

[0233]

[0234]

[0235] HOMA: Homeostatic Model Assessment, HOMA index = insulin (fasting, μU / ml) x glucose (fasting, mg / dl) / 405, HOMA index > 2 is an indicator of insulin resistance.

[0236] Clinical results:

[0237] A significant reduction of insulin resistance and infection rate was observed. The typical skin discoloration of the Sjogren syndrome was improved. Hair loss stopped. EBV infected B cells in the patient were significantly reduced Figure 7 ).

[0238] Example 7 (Rheumatoid arthritis, insulin resistance)

[0239] A 44 year old male, positive for EBV test, suffers from rheumatoid polyarthritis, metabolic syndrome and insulin resistance.

[0240] In a detailed EBV diagnosis, for example, a medium frequency of EBER-1+2 CISH positive B cells (25-50%) can be determined in the patient's enriched B cell fraction. In addition, an abnormally high number (38) of gp350 positive B cells was detected, indicating that EBV reactivation is active in this patient.

[0241] Due to this diagnosis and unmet medical need, the patient decided to participate in a compassionate use treatment with a research anti-EBV therapeutic vaccine, as described in Example 2.

[0242] A cell preparation similar to Examples 1 and 2 was generated ex vivo and applied subcutaneously back to the patient (August 18, 2015).

[0243] For the booster application, the same procedure was repeated (September 15, 2015).

[0244] The following diagnostic parameters are evaluated before, during and after the treatment:

[0245] Immunological results:

[0246] Humoral anti-EBV response

[0247]

[0248] Enriched B cells (CD20, 2.5 x 10e5) stained with anti-gp350 antibody APAAP technique

[0249]

[0250]

[0251] HOMA: Homeostatic Model Assessment, HOMA index = insulin (fasting, μU / ml) x glucose (fasting, mg / dl) / 405, HOMA index > 2 is an indicator of insulin resistance.

[0252] RF, rheumatoid factor: autoantibody of IgM subclass, binds to the constant Fc region of antibodies.

[0253] ANA-IFT: anti-nuclear antibodies, immunofluorescence test, indicator of autoimmune diseases such as RA.

[0254] Clinical results:

[0255] Seven weeks after the boost of the immunity (15 September 2015), the symptoms of rheumatoid arthritis were significantly improved, which was consistent with the decrease of the inflammatory cytokines IL-5, IL-22 and INF-γ and with the laboratory values related to rheumatism (RF, ANA-IFT). Since September 2015 (at least six months of follow-up), no analgesics were required. The swelling of the hand joints was significantly reduced. The insulin resistance was normalized. During 2016, no relapse of rheumatoid arthritis was observed and the Hashimoto's syndrome and eczema (rosacea) remained stable. The EBV-infected B cells in the patient were significantly reduced ( Figure 8 and 9 ) as the treatment progressed.

[0256] Example 8 (Type 1 diabetes)

[0257] The 9-year-old girl who tested positive for EBV had insulin resistance of type 1 diabetes.

[0258] In a detailed EBV diagnosis, a high frequency of EBNA-1+2 CISH-positive B cells (25-50%) can be determined, for example, in the enriched B cell fraction of the patient.

[0259] Due to this diagnosis and unmet medical need, the patient decided to participate in a compassionate use treatment with a research anti-EBV therapeutic vaccine as described in Example 2.

[0260] Cellular preparations similar to Example 1 and 2 were generated ex vivo and applied subcutaneously back to the patient (November 11, 2015).

[0261] Enriched B cells (CD20, 2.5 x 10e5) were stained with anti-gp350 antibody APAAP technique

[0262]

[0263] Clinical results:

[0264] After treatment, growth as well as overall energy and sleep quality improved. HBA1 c values remained stable and no severe infections were observed during follow-up. EBV infected B cells in the patient significantly decreased Figure 10 ).

[0265] Example 9 (vaginal lichen)

[0266] A 65 year old female with positive EBV test suffers from vaginal lichen, chronic vaginal infection, chronic vaginal pain, chronic fatigue and poor concentration.

[0267] In a detailed EBV diagnosis, e.g. a high frequency of EBER-1+2 CISH positive B cells (50-75%) can be determined in the patient's enriched B cell fraction.

[0268] Due to this diagnosis and unmet medical need, the patient decided to participate in a compassionate use treatment with a research anti-EBV therapeutic vaccine as described in Example 2.

[0269] Cellular preparations similar to Example 1 and 2 were generated ex vivo and applied subcutaneously back to the patient on October 22, 2015.

[0270] For the booster application, the same steps were repeated (November 19, 2015).

[0271] Before, during and after treatment the following diagnostic parameters were evaluated:

[0272]

[0273] HOMA: Homeostatic Model Assessment, HOMA index = insulin (fasting, μU / ml) x glucose (fasting, mg / dl) / 405, HOMA index > 2 is an indicator for insulin resistance.

[0274] RF, rheumatoid factor: autoantibody of IgM subclass, binds to the constant Fc region of antibodies.

[0275] ANA-IFT: antinuclear antibodies, immunofluorescence test, marker for autoimmune diseases like RA.

[0276] Clinical results:

[0277] In October 2015, the patient developed chronic vaginal infections, chronic vaginal pain, chronic fatigue and poor concentration. During the post-treatment check-ups in 01 / 2016, 05 / 2016 and 10 / 2016, the patient's condition improved continuously, which was also confirmed by the laboratory parameters, since a reduction of B-cell EBV load as well as cytokine titers was observed. In the last check-up in June 2017, the B-cell EBV load was still < 25% and the patient was again back to normalization of energy, without infections, and a reduction of vaginal lichens and without vaginal pain. With the progression of the treatment, the EBV-infected B-cells in the patient significantly decreased Figure 11 and 12 ).

[0278] Example 10 (chronic cystitis and prostatitis)

[0279] A 65-year-old male, positive for EBV testing, suffers from chronic cystitis and prostatitis, depression, fatigue, cough and insulin resistance.

[0280] In a detailed EBV diagnosis, e.g. a high frequency of EBER-1+2 CISH positive B-cells (> 75%) can be determined in the patient's enriched B-cell fraction.

[0281] Due to this diagnosis and unmet medical need, the patient decided to participate in a compassionate use treatment with a research-based anti-EBV therapeutic vaccine, as described in Example 2.

[0282] A cell preparation similar to Example 1 and 2 was generated ex vivo and applied subcutaneously back to the patient on 10 / 16 / 15.

[0283] For the booster application, the same steps were repeated (27 / 04 / 17).

[0284] The following diagnostic parameters were evaluated before, during and after the treatment:

[0285] Enriched B-cells (CD20, 2.5 x 10e5) were stained with anti-gp350 antibody APAAP technique

[0286]

[0287] Clinical results:

[0288] During the visit in May 2016 after treatment, an improvement of attention and concentration was observed. Prostatitis stopped, but reoccurred once in March 2016, which could be treated with antibiotics. Cystitis as well as general infection rate improved. Although the antidepressant could be reduced from 3 different antidepressants to one antidepressant, barbiturates could also be reduced. A reduction of fibromyalgia was also observed. With the progression of the treatment, the B cells of the EBV infection in the patient significantly decreased Figure 13 and 14 ).

[0289] Example 11 (Multiple Sclerosis)

[0290] A 40 year old female, positive for EBV detection, suffered from multiple sclerosis (MS). In a detailed EBV diagnosis, e.g. a high frequency of EBER-1+2 CISH positive B cells (>75%) could be determined in a patient's enriched B cell fraction.

[0291] Due to this diagnosis and unmet medical need, the patient decided to participate in a compassionate use treatment with a research-based anti-EBV therapeutic vaccine, as described in Example 2.

[0292] Cellular preparations similar to Example 1 and 2 were generated ex vivo and applied subcutaneously back to the patient (3rd December 2015).

[0293] For the booster application, the same procedure was repeated (6th November 2016).

[0294] The following diagnostic parameters were evaluated before, during and after treatment:

[0295] Enriched B cells (CD20, 2.5 x 10e5) were stained with anti-gp350 antibody APAAP technique

[0296]

[0297] Clinical outcome

[0298] The patient has a progressive multiple sclerosis, diagnosed since mid-2011 by MRI and lumbar puncture, with symptoms of heavy legs, numb feet, walking and balance difficulties, and also associated with shooting pains in both legs. The patient is treated with Avonex 30 mg cortisone. In early 2012, the patient has a progression, with more active MS lesions C4 / 5 (cervical) found in MRI. The treatment is then changed to interferon. The patient has symptoms of attention problems, sleep disorders, fatigue, increased numbness in the right leg and foot, more unbalanced walking. In December 2013, with further progression and development of depression, multiple eczema (first diagnosis) appears, with more progressive MS lesions C4 / 5 found in MRI. The treatment is changed to refib, cortisone. The patient's symptoms are less than 1 km of walking due to increased pain, and must walk with help, right arm weakness and attention problems. In March 2014, progression of the disease with new lesions in the cerebellum area (brain) and thoracic spine (T2 = precise localization: second thoracic vertebra) found in MRI, as well as new progression of lesions C4 / 5. There is no longer a standard treatment available. An experiment with fampridine is made, with 2 dendritic cell treatments.

[0299] At the beginning of the experimental treatment, the patient has symptoms of dizziness, increased fatigue, heavy legs, more numbness on the right side and sleep disorders.

[0300] In July 2014, the disease progression is found in MRI with lesions C4 / 5, increased numbness in the brain, neurodegeneration and demyelination processes. A therapy with light penetration and anti-inflammatory infusion is started. The patient's symptoms are loss of control of the right leg, increased fatigue, increased memory and attention problems, more serious sleep problems.

[0301] February 2015: stable disease C4 / 5 found in MRI, lower T2 activity, normal cerebellum, less eczema. Anti-inflammatory treatment with herbal extracts (turmeric) and omega 3-fatty acids is ongoing. The patient shows symptoms of improved walking, less numbness, better sleep and more energy.

[0302] November 2015: progression found in MRI in C4 / 5, progression T2. The patient has symptoms of increased fatigue, increased numbness in the right foot, decreased sensitivity in the right arm, tingling in the right hand fingers, dizziness and dizziness. First therapeutic EBV vaccination (compassionate use) started in early December 2015.

[0303] June 2016: stable disease C4 / 5 and T2 found in MRI. The patient shows symptoms of further improved numbness, more balanced, no dizziness, no dizziness, more energy.

[0304] October 2016: C4 / 5 stable disease, T2 mild progression found on MRI. Patient showed symptoms of increased foot and leg numbness, no rashes, more energy, improved sleep.

[0305] November 6, 2016: Second therapeutic EBV vaccination started.

[0306] March 2017: C4 / 5 stable on MRI. Patient had symptoms of less numbness.

[0307] September 2017: C4 / 5 inactive lesion found on MRI, T2: no lesion seen.

[0308] April 2018: MRI: Patient showed stable appearance.

[0309] With ongoing EBV vaccination therapy, EBV infected B cells in the patient significantly decreased Figure 15 and 16 ).

[0310] Example 12 (migraine, alopecia vulgaris, acne, weight gain, chronic fatigue syndrome, poor concentration, poor memory)

[0311] A female patient (date of birth: January 28, 1997) who tested positive for EBV reactivation (high frequency of EBER-1+2 CISH positive B cells) showed autoimmune symptoms including migraine, alopecia vulgaris, acne, weight gain, chronic fatigue syndrome, poor concentration, and poor memory.

[0312] In August 2015, a cell preparation similar to Examples 1 and 2 was generated ex vivo and applied subcutaneously back to the patient.

[0313] For the booster application, the same procedure was repeated in October 2015.

[0314]

[0315]

[0316] Interleukin-17 <1 pg / ml 8.2 0.1 Interleukin-22 <1 pg / ml 13.8 0.1 Interleukin-23 <1 pg / ml - -

[0317] Clinical results:

[0318] Since June 2016, the patient has normal hair growth, weight loss of 8 kg, normalized attention and better energy. No more headaches were observed. The improvement of the clinical outcome is related to the decrease of EBV positive B cells (CISH analysis showed a decrease from 50-75% to <25% category), the decrease of immune complexes (CIC-IgM, CIC-IgG and CIC-C3) and the normalization of inflammatory cytokine values of IL-8, IL-17 and IL-22.

[0319] Example 13 (Metabolic syndrome, weight gain, poor memory, reflux, hair loss)

[0320] A female patient (date of birth: 09 October 1989) who tested positive for EBV reactivation (high frequency of EBER-1+2 CISH positive B cells) showed autoimmune symptoms including metabolic syndrome, weight gain, poor memory, reflux and hair loss.

[0321] In August 2015, a cell preparation similar to Examples 1 and 2 was generated ex vivo and applied subcutaneously back to the patient.

[0322] For the booster application, the same procedure was repeated in October 2015.

[0323]

[0324]

[0325] HOMA <2 2.3 1.2 Interleukin-17 <1 pg / ml - - Interleukin-22 <1 pg / ml Interleukin-23 <1 pg / ml 160 75

[0326] HOMA: Homeostatic Model Assessment, HOMA index = insulin (fasting, μU / ml) x glucose (fasting, mg / dl) / 405, HOMA index > 2 is an indicator for insulin resistance

[0327] ANA-IFT: Anti-nuclear antibodies, immunofluorescence test, indicator for autoimmune diseases such as RA

[0328] Clinical results:

[0329] Since 2016, the patient's energy improved and lost 5 kg of weight. Since 2018, the patient lost 10 kg of weight, hair regrew normally, memory normalized and no infections. The improvement of the clinical outcome is related to the decrease of EBV positive B cells (CISH analysis showed a decrease from 50-75% to <25% category), the decrease of immune complexes (CIC-IgM, CIC-IgG and CIC-C3), the normalization or decrease of inflammatory cytokine values of IL-8 and IL-23 and the normalization of HOMA values.

[0330] Example 14 (chronic herpes zoster, chronic prostatitis, chronic fatigue syndrome)

[0331] A male patient (date of birth: June 30, 1957) positive for EBV reactivation test (high frequency of EBER-1+2 CISH positive B cells) showed chronic herpes zoster, chronic prostatitis and chronic fatigue syndrome and was treated with various antibiotics.

[0332] Cell preparations similar to Examples 1 and 2 were generated ex vivo and applied subcutaneously back to the patient on November 6, 2015.

[0333] For the booster application, the same procedure was repeated at the end of November 2015.

[0334]

[0335] ANA-IFT: Anti-nuclear antibodies, immunofluorescence test, marker for autoimmune diseases, such as RA

[0336] Clinical results:

[0337] Since 2016, the patient has no herpes zoster, no fatigue, less antibiotics and less frequent prostatitis infections. The improvement of the clinical outcome is associated with a reduction of EBV positive B cells (CISH analysis shows a reduction from 50-75% to 25-50% class), a reduction of immune complexes (CIC-IgM and CIC-IgG), anti-nuclear antibodies and PSA to normal values.

[0338] Example 15 (chronic hepatitis (non-HBV, HCV), chronic pancreatitis, chronic fatigue syndrome, diarrhea / IBS, chronic bronchitis)

[0339] A male patient (date of birth: June 23, 1965) positive for EBV reactivation test (high frequency of EBER-1+2 CISH positive B cells) showed autoimmune symptoms and EBV-induced hepatitis including chronic hepatitis (non-HBV, HCV), chronic pancreatitis, chronic fatigue syndrome, diarrhea / IBS and chronic bronchitis.

[0340] Cell preparations similar to Examples 1 and 2 were generated ex vivo and applied subcutaneously back to the patient on November 6, 2015.

[0341] For the booster application, the same procedure was repeated, with the first booster in November 2015 and the second booster in June 2017.

[0342]

[0343] ANA-IFT: Anti-nuclear antibodies, immunofluorescence test, marker for autoimmune diseases, e.g. RA

[0344] Clinical results:

[0345] Since 2018, the patient has no fatigue and no diarrhea, IBS (irritable bowel syndrome) is normalized, infection frequency is reduced. Hepatitis is improved and liver enzymes are reduced to normal values. Furthermore, the improved clinical outcome is associated with a reduction of EBV-positive B cells (CISH analysis shows a reduction from 75% to <25% category), a reduction of immune complexes (CIC-IgM, CIC-IgG and CIC-C3), a reduction of anti-nuclear antibodies and a reduction of the inflammatory cytokines IL-6 and IL-8, especially after the second boost with Trivacc.

[0346] Example 16 (chronic fatigue syndrome, diarrhea / IBS colitis, chronic flue-like symptoms, Hashimoto's disease)

[0347] A male patient (date of birth: August 1, 1986) who tested positive for EBV reactivation (high frequency of EBER-1+2 CISH-positive B cells) showed autoimmune symptoms including chronic fatigue syndrome, diarrhea / IBS colitis, chronic flue-like symptoms, and Hashimoto's disease.

[0348] In October 2015, a cell preparation similar to Examples 1 and 2 was generated ex vivo and applied subcutaneously back to the patient.

[0349] For the boost application, the same procedure was repeated in November 2015.

[0350]

[0351] ANA-IFT: Anti-nuclear antibodies, immunofluorescence test, marker for autoimmune diseases, e.g. RA

[0352] Clinical results: Clinical results:

[0353] Since 2018, the patient has no fatigue and no diarrhea, IBS (irritable bowel syndrome) is normalized, infection frequency is reduced. Hepatitis is improved and liver enzymes are reduced to normal values. Furthermore, the improved clinical outcome is associated with a reduction of EBV-positive B cells (CISH analysis shows a reduction from 75% to <25% category), a reduction of immune complexes (CIC-IgM, CIC-IgG and CIC-C3), a reduction of anti-nuclear antibodies and a reduction of the inflammatory cytokines IL-6 and IL-8, especially after the second boost with Trivacc.

[0354] Example 17 (Hashimoto's disease, EBV-autoimmune hepatitis, EBV-autoimmune pancreatitis with insulin resistance)

[0355] The patient (DOB: 14 December 1978) who tested positive for EBV reactivation (high frequency of EBER-1+2 CISH positive B cells) showed autoimmune symptoms including Hashimoto’s disease, EBV-autoimmune hepatitis and EBV-autoimmune pancreatitis with insulin resistance.

[0356] The patient’s history is shown as follows:

[0357] 1992 mononucleosis

[0358] 1998 Campylobacter, measles, rubella reactivation

[0359] 2003 shingles

[0360] July 2014 large lobar pneumonia

[0361] July 2015 large lobar pneumonia

[0362] October 2015 viral induced pericarditis (EBV and herpes 2)

[0363] December 2015 influenza B, EBV positive in B cells (CISH)

[0364] January 2016 diabetes, hyperosmolarity

[0365] April 2016 pleural effusion, enterovirus and coxsackie virus negative

[0366] May 2018 IBS (irritable bowel syndrome), esophageal varices and bleeding

[0367] July 2018 EBV based hepatitis and pancreatitis, diabetes / insulin resistance more severe, first therapeutic EBV vaccination

[0368] September-December 2018 treatment with dulaglutide, but stopped due to side effects

[0369] January 2019 HOMA value significantly improved, no diabetes, IBS improved, less diarrhea, recurrence of hepatitis, second therapeutic EBV vaccination

[0370] March 2019 In general, an improvement of the clinical situation can be observed after the first and second therapeutic EBV vaccination (Trivacc). Thus, an improvement of the clinical situation and laboratory values of Hashimoto's disease (normalization of anti-TPO values), insulin resistance (HOMA decreased from 20.8 to 4.0) and hepatitis (improvement of liver values after the second EBV vaccination for GOT, GPT, GGT and bilirubin, etc.). At the same time, according to the PCR data, the viral load of EBV is significantly reduced (PCR cycle increased from 31.08 to 32.27, indicating a lower viral load). This result is supported by a reduction of the number of EBV-positive B cells in the peripheral blood (CISH results decreased from > 75% to the 25-50% category after the second vaccination). Furthermore, the immune complexes in the blood, which are a marker of autoimmune responses, are also reduced and reach the background values of CIC-IgA, CIC-IgG, CIC-C3. The antinuclear antibodies (ANA-IFT) are also reduced to background levels.

[0371]

[0372]

[0373]

[0374] HOMA: Homeostatic Model Assessment, HOMA index = insulin (fasting, μU / ml) x glucose (fasting, mg / dl) / 405, HOMA index > 2 is an indicator of insulin resistance

[0375]

[0376] PCR - virus

[0377]

Claims

1. The use of the isolated trifunctional bispecific antibody in the preparation of a medicament for treating patients with diseases and / or conditions associated with reactivation of Epstein-Barr virus (EBV) in at least B cells and potentially other susceptible cells, said use comprising: Provide the patient with an autologous cell preparation containing enriched B cells; The enriched B cells are incubated with a trifunctional bispecific antibody for a period of time sufficient to establish a physical interaction between the trifunctional bispecific antibody and the enriched B cells to obtain an incubation mixture; the incubation mixture obtained after incubation is transferred to the same patient, wherein the trifunctional bispecific antibody recruits B cells and T cells, and also recruits Fc receptor-positive cells, and comprises: (a) Binding to the first binding arm of a B cell via a B cell surface antigen; (b) Binding to the second binding arm of T cells via T cell surface antigens; (c) The Fc portion that binds to Fc receptor-positive cells. The antibody mentioned therein is an anti-CD3 x anti-CD20 bispecific antibody, and The diseases and / or conditions associated with the reactivation of EBV in B cells are selected from one or more autoimmune diseases in the group consisting of rheumatoid arthritis, Hashimoto's disease, type 1 diabetes, and multiple sclerosis, or The diseases and / or conditions associated with the reactivation of EBV in B cells are selected from one or more chronic inflammatory diseases comprising the group consisting of chronic prostatitis, chronic cystitis, non-alcoholic chronic hepatitis, irritable bowel syndrome, chronic neuroinflammation, and metabolic syndrome, or The diseases and / or conditions associated with the reactivation of EBV in B cells are selected from one or more diseases and conditions in the group consisting of Sjögren's syndrome, myasthenia gravis, Crohn's disease, vitiligo, type 2 diabetes, chronic ductitis, vaginal lichen, chronic pancreatitis, chronic bronchitis and chronic smoker symptoms, chronic fatigue syndrome, chronic dry cough, chronic rhinitis, night sweats, sleep disorders; insomnia, polyneuropathy, edema in the fingers, toes and face, endometriosis, ovarian cysts, menstrual disorders, hair loss, memory problems, dry eyes and dry mouth, migraine, acne, weight gain, poor attention, insulin resistance, diarrhea and chronic herpes zoster.

2. The use according to claim 1, wherein the antibody further comprises an autologous cell preparation of peripheral blood mononuclear cells (PBMCs) from the same patient, wherein the PBMCs are added to the enriched B cells and the trifunctional bispecific antibody incubation mixture for a period of time sufficient to establish a physical interaction between the PBMCs and the trifunctional bispecific antibody before the incubation mixture is transferred to the same patient, wherein the period of time is from 1 min to 60 min.

3. The use according to claim 1 or 2, wherein the incubation period for establishing the physical interaction between the trifunctional bispecific antibody and the enriched B cells is from 1 min to 60 min.

4. The use according to claim 1 or 2, wherein the antibody is administered in an amount from 0.1 μg to 100 μg.

5. The use according to claim 1 or 2, wherein the antibody is a rat / mouse bispecific antibody.

6. The use according to claim 1 or 2, wherein the antibody contains a binding site in its Fc portion for Fcγ receptor types I, IIa and / or III.

7. The use according to claim 1 or 2, wherein the antibodies are capable of binding to monocytes, macrophages, dendritic cells, natural killer cells and / or activated neutrophils via their Fcγ receptors type I, IIa and / or III.

8. The use according to claim 1 or 2, wherein the antibody is selected from at least one member of the group consisting of the following isotype combinations in its Fc portion: Rat-IgG2b / Mouse-IgG2a; Rat-IgG2b / Mouse-IgG2b; Rat-IgG2b / human-IgG1; Human-IgG1 / Human-IgG1-[hinge]-Human-IgG3*--[CH2-CH3], where * = Caucasian allotype G3m(b+g) = does not bind to protein A; Mouse-[VH-CH1,VL-CL]-human-IgG1 / rat-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3], where * = Caucasian allotype G3m(b+g) = does not bind to protein A; and mouse-[VH-VL]-human–[CH1-CL]-human-IgG1 / rat-[VH-CH1,VL-CL]-human-IgG1-[hinge]-human-IgG3*-[CH2-CH3], where * = Caucasian allotype G3m(b+g) = does not bind to protein A.

9. The use according to claim 1 or 2, wherein the medicament comprises a pharmaceutically acceptable carrier and / or excipient.

10. The use according to claim 9, wherein the drug is in the form of a "component kit".

11. The use according to claim 1 or 2, wherein the disease and / or condition associated with the reactivation of EBV in B cells is common alopecia.

12. An in vitro method for preparing a pharmaceutical composition for treating a patient with a disease and / or condition associated with reactivation of EBV in at least B cells and potentially other susceptible cells, the method comprising: Provide the patient with an autologous cell preparation containing enriched B cells; The enriched B cells are incubated with the trifunctional bispecific antibody for a period of time sufficient to establish a physical interaction between the trifunctional bispecific antibody and the enriched B cells to obtain an incubation mixture, wherein the trifunctional bispecific antibody comprises: (a) Binding to the first binding arm of a B cell via a B cell surface antigen; (b) Binding to the second binding arm of T cells via T cell surface antigens; (c) The Fc portion that binds to Fc receptor-positive cells. The antibody mentioned therein is an anti-CD3 x anti-CD20 bispecific antibody, and The diseases and / or conditions associated with the reactivation of EBV in B cells are selected from one or more autoimmune diseases in the group consisting of rheumatoid arthritis, Hashimoto's disease, type 1 diabetes, and multiple sclerosis, or The diseases and / or conditions associated with the reactivation of EBV in B cells are selected from one or more chronic inflammatory diseases in the group consisting of chronic prostatitis, chronic cystitis, non-alcoholic chronic hepatitis, irritable bowel syndrome (IBS), chronic neuroinflammatory disease, and metabolic syndrome; or The diseases and / or conditions associated with the reactivation of EBV in B cells are selected from Sjögren's syndrome, myasthenia gravis, Crohn's disease, vitiligo, type 2 diabetes, chronic ductitis, vaginal lichen, chronic pancreatitis, chronic bronchitis and chronic smoker symptoms, chronic fatigue syndrome, chronic dry cough, chronic rhinitis, night sweats, and sleep disorders. Insomnia, polyneuropathy, swelling in the fingers, toes and face, endometriosis, ovarian cysts, menstrual irregularities, hair loss, memory problems, dry eyes and mouth, migraines, acne, weight gain, poor concentration, insulin resistance, diarrhea and chronic herpes zoster are one or more diseases and conditions.

13. The in vitro method for preparing a pharmaceutical composition according to claim 12, the method further comprising an autologous cell preparation of monocytes from the same patient's PBMCs, wherein the PBMCs are added to an incubation mixture of the enriched B cells and the trifunctional bispecific antibody for a duration sufficient to establish a physical interaction between the PBMCs and the trifunctional bispecific antibody, wherein the time is from 1 min to 60 min.

14. The in vitro method for preparing a pharmaceutical composition according to claim 12 or 13, wherein the disease and / or condition associated with the reactivation of EBV in B cells is common alopecia.

15. The use of an incubation mixture comprising a trifunctional bispecific antibody and an autologous cell preparation of enriched B cells from a patient in the preparation of a medicament for treating patients with at least B cells and potentially other susceptible cells associated with Epstein-Barr virus (EBV) reactivation, said use comprising: Provide the patient with an autologous cell preparation containing enriched B cells; The enriched B cells are incubated with the trifunctional bispecific antibody for a period of time sufficient to establish a physical interaction between the trifunctional bispecific antibody and the enriched B cells to obtain the incubation mixture; the incubation mixture obtained after incubation is transferred to the same patient, wherein the trifunctional bispecific antibody comprises: (a) Binding to the first binding arm of a B cell via a B cell surface antigen; (b) Binding to the second binding arm of T cells via T cell surface antigens; (c) The Fc portion that binds to Fc receptor-positive cells. The antibody mentioned therein is an anti-CD3 x anti-CD20 bispecific antibody, and The diseases and / or conditions associated with the reactivation of EBV in B cells are selected from one or more autoimmune diseases in the group consisting of rheumatoid arthritis, Hashimoto's disease, type 1 diabetes, and multiple sclerosis, or The diseases and / or conditions associated with the reactivation of EBV in B cells are selected from one or more chronic inflammatory diseases comprising the group consisting of chronic prostatitis, chronic cystitis, non-alcoholic chronic hepatitis, irritable bowel syndrome (IBS), chronic neuroinflammatory disease, and metabolic syndrome. The diseases and / or conditions associated with the reactivation of EBV in B cells are selected from one or more diseases and conditions in the group consisting of Sjögren's syndrome, myasthenia gravis, Crohn's disease, vitiligo, type 2 diabetes, chronic ductitis, vaginal lichen, chronic pancreatitis, chronic bronchitis and chronic smoker symptoms, chronic fatigue syndrome, chronic dry cough, chronic rhinitis, night sweats, sleep disorders; insomnia, polyneuropathy, edema in the fingers, toes and face, endometriosis, ovarian cysts, menstrual disorders, hair loss, memory problems, dry eyes and dry mouth, migraine, acne, weight gain, poor attention, insulin resistance, diarrhea and chronic herpes zoster.

16. The use according to claim 15, wherein the disease and / or condition associated with the reactivation of EBV in B cells is common alopecia.

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