Biological binding molecules

Abstract

To provide alternative concepts and active agents, such as alternative antibodies for diagnosis, prevention, and / or treatment of malignant B-cell neoplasms.SOLUTION: The present invention provides a composition for diagnosis, prevention and / or treatment of cancer disease, where the composition comprises a biological binding molecule, which is an antibody or a fragment having its antigen-binding site, containing CDR1, CDR2, and CDR3 of the heavy chain variable region with specific sequences, and CDR1, CDR2, and CDR3 of the light chain variable region with specific sequences.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to the field of production, identification and selection of antibodies or fragments thereof and their use in the field of prevention and treatment of cancer diseases, particularly malignant B-cell neoplasms, and associated diagnostics.

[0002] Malignant B-cell neoplasms generally represent malignant disorders of the hematopoietic or lymphatic systems. They include clinical manifestations such as leukemia, broadly classified as cancer. Leukemia is characterized by a massive increase in the formation of dysfunctional white blood cell precursors, also known as leukemic cells. These cells spread to the bone marrow, where they replace normal blood formation, proliferating and accumulating in the peripheral blood. They can infiltrate the liver, spleen, lymph nodes, and other organs, thereby impairing their function. Impaired blood formation can lead to a decrease in normal blood components, resulting in anemia due to a lack of oxygen-carrying red blood cells, hemostatic platelets, and mature, functional white blood cells.

[0003] Depending on the course of the disease, a distinction is made between acute and chronic leukemia. Acute leukemia is a life-threatening disease that, if left untreated, leads to death within weeks to months. Chronic leukemia, on the other hand, usually progresses over several years, often with symptoms subsiding in the early stages.

[0004] The most important forms of leukemia are: Acute myeloid leukemia (AML) Chronic myeloid leukemia (CML) Acute lymphoblastic leukemia (ALL) Chronic lymphocytic leukemia (CLL)

[0005] Leukemia is usually treated with a range of chemotherapy. In this regard, more recent treatments have increasingly used monoclonal antibodies, such as GA101 (obinutuzumab), which acts as a CD20 antibody, similar to rituximab and ofatumumab, and is used to treat chronic lymphocytic leukemia (CLL). The use of these antibodies can extend the remission-free period by approximately 10 months.

[0006] Other malignancies of the hematopoietic or lymphatic system (malignant B-cell neoplasms) affect lymphomas, for example, Hodgkin's lymphoma and the B-cell variants of non-Hodgkin's lymphoma.

[0007] Once antibodies to a receptor have been generated, animals are typically immunized with the receptor (purified, cloned, or as a peptide fragment) to generate hybridoma cells. These hybridoma cells produce antibodies that are tested in cell lines using ELISA or expressed receptors. Traditionally, established cell lines are used for this purpose because they can only be easily cultured. In this case, antibodies can be generated that bind relatively specifically to a particular receptor type (e.g., anti-IgG1, anti-IgE). However, this often leads to cross-reactivity with other receptors or other epitopes.

[0008] In diagnostic or therapeutic applications of BCR antibodies, it is generally not sufficient to use only one antibody against BCR in most cases, because such a broad spectrum of antibodies can lead to false-positive results or cause significant side effects. Instead, it would be desirable to provide an antibody that selectively binds to a receptor having a light chain with the epitope VL3-21 according to SEQ ID NO: 18. This light chain epitope is overexpressed on neoplastic B cells. Such an antibody is not known in the prior art, and no method exists for its production or selection.

[0009] Prior art treatments for the treatment of leukemia are very burdensome for patients. Generally speaking, it can be said that the undesirable side effects of treatment and the often insufficient efficacy of drugs lead to the high mortality rate of this disease. This is because not only tumor cells but also healthy cells of the immune system are damaged. Moreover, in many cases, there is no cure, and only a certain period of disease progression without remission occurs. Therefore, the use of diagnostic means and methods capable of distinguishing and detecting specific forms of malignant B-cell neoplasms is important for identifying and selecting patients to be treated and for developing individual treatment plans.

[0010] It is therefore an object of the present invention to provide alternative concepts and agents, such as alternative antibodies, in particular for diagnostic, prophylactic and / or therapeutic use, which overcome the existing problems of the prior art. Advantageously, the present invention is also suitable for detecting the presence of surface structures suitable for therapeutic use of antibodies ("companion diagnostics").

[0011] Before discussing individual aspects of the present invention in detail, the meaning of relevant terms used within the present specification will be clarified.

[0012] The term "neoplasm" as used herein generally refers to the formation of new bodily tissue. When this is a pathological or malignant condition, it refers to a malignant neoplasm. That is, a malignant B-cell neoplasm is the malignant, uncontrolled formation of new tissue of B cells, and this term applies equally to all B-cell-related cancer diseases, such as leukemia and B-cell lymphoma.

[0013] A "neocidal domain" is capable of killing a neoplasm as a result of direct or indirect action. When a particular antibody is used therapeutically, an effect beyond that of the antibody or its fragment can be achieved by conjugating a molecule to the antibody or its functional fragment, or to another biological binding molecule comprising the antibody or its fragment. Such a molecule can be selected from the group consisting of, for example, an immunotoxin, a cytokine, a chelating agent, a radioisotope, and combinations thereof.

[0014] The term "biological binding molecule" as used herein refers to, but is not limited to, antibodies, including, for example, fusion proteins. Advantageously and therefore preferably, such antibodies are selected from the group consisting of IgG antibodies, IgM antibodies, humanized IgG antibodies, and human antibodies into which an epitope recognition sequence is inserted. Such binding molecules may be provided in the form of functional fragments of whole antibodies, such as Fab fragments. Furthermore, binding molecules may further comprise regions that, for example, lead to the killing / death of neoplasms and, accordingly, have the function of immunotoxins and / or immunocytokines. In particular, such binding molecules may be membrane- or cell-bound. Such membrane-bound binding molecules are, for example, chimeric antigen receptors of CAR-T cells.

[0015] Furthermore, binding molecules may also contain further regions or additional entities, the use of which is advantageous in the field of diagnostic applications. These are fluorescent dyes (e.g., FITC, R-phycoerythrin (R-PE), allophycocyanin (APC)) or alternatively biotin, as well as other substances known to those skilled in the art, for use in flow cytometry. Binding molecules can also be used together with substrate-converting enzymes (e.g., HRP) in immunohistochemistry processes. Furthermore, for diagnostic purposes, fusion proteins can also be provided, in which a fluorescent protein, such as green fluorescent protein (GFP), is attached to the Fc portion of an antibody for detection.

[0016] The function of the B cell receptor complex (BCR) on the surface of B cells is to recognize and bind to pathogens. This binding leads to a conformational change in the BCR, which triggers a signaling cascade that ultimately leads to B cell activation. The BCR is highly diverse and is formed in maturing B cells.

[0017] In humans and some other mammals, B cell development occurs in the bone marrow or fetal liver. Signals necessary for the developmental program are received by lymphocytes emerging from so-called stromal cells. The formation of a functional B cell receptor (a membrane-bound form of an antibody) is crucial for B cell development. Only through this antigen receptor can mature B cells subsequently recognize foreign antigens and bind to hostile structures by forming the corresponding antibodies. The receptor's antigen specificity is determined by the joining of specific gene segments. These segments are called V-, D-, and J-segments, a process called V(D)J rearrangement. These segments rearrange to form the antigen-binding portion of the B cell receptor. The entire receptor consists of two identical light protein chains and two identical heavy protein chains, each linked by a disulfide bridge.

[0018] Each light chain consists of a variable domain and a constant domain. The variable region plays a crucial role in antigen recognition, while the constant region determines the five immunoglobulin classes and, in the case of T cell receptors, is responsible for membrane anchoring. Light chains are constructed by somatic recombination from pools of V and J genes and the constant chains lambda or kappa. Heavy chains are made up of three parts (V, D, and J) that make up the variable region. While heavy chains have traditionally been the focus of research and have been extensively studied, light chains have received little attention. Two loci for light chains are known: the kappa locus with 40 V gene segments and the lambda locus with 30 V gene segments. In CLL, certain combinations occur predominantly in the corresponding B cells (Stamatopoulus et al. 2005 (BLOOD Vol. 106, Number 10)). In particular, analysis of B cells from CLL patients revealed that the V chain VL3-21 is present in a surprisingly large number of patient cells with autonomously active BCRs. Three variants of the V chain VL3-21 are known to exist, and these variants differ from each other by a maximum of two amino acids and can therefore be used with equal effect in the present invention.

[0019] The large repertoire of immunoglobulin and T-cell receptor specificities, which would exceed the size of the genome if each molecule contained a separate gene, is made possible in particular by the existence of multiple copies of individual gene segments (V, D, J) that, prior to rearrangement, can be combined arbitrarily with each other like a sort of combination lock during lymphocyte maturation.

[0020] In VDJ rearrangement, the V, D, and J segments of the B cell receptor heavy chain are joined first, followed by the V and J segments of the receptor light chain. Only successful gene rearrangement, referred to as productive gene rearrangement, allows cells to progress to the next developmental stage.

[0021] During their maturation in the bone marrow, B cells that react to the body's own antigens usually die by apoptosis. In the blood of healthy people, small numbers of autoreactive cells, particularly against thyroglobulin and / or collagen, can be detected (Abul K. Abbas: Diseases of Immunity in Vinay Kumar, Abul K. Abbas, Nelson Fausto: Robbins and Cotran - Pathologic Basis of Disease; 7th edition; Philadelphia 2005, p. 224f).

[0022] Because the process of generating such BCRs is based on the random assembly of gene segments, newly formed BCRs may recognize unnecessary biological structures and thus become "permanently activated." Various protective mechanisms exist to prevent the formation of such "permanently active or activated" BCRs. However, when these are overcome based on pathological changes in developing B cells, malignant or even autoimmune phenotypes can result.

[0023] In contrast, "autonomously active" or "autonomously activated" BCRs are a special type of constitutively active BCR. While conventional activation begins with an external antigen (see above), autonomously activated BCRs result from interactions with membrane structures on the surface of the same cell. Regarding the clinical manifestations of CLL, interactions that trigger autonomous activation between BCRs located adjacent to each other on the surface of the same cell have been demonstrated (M. Duehren-von Minden et al.; Nature 2012). Another example of an autonomously active BCR is the pre-BCR, which is expressed as a developmental checkpoint throughout B cell development. On the other hand, in addition to interactions between adjacent receptors (BCR:BCR), interactions between receptors and membrane proteins (BCR:membrane proteins) also result in autonomously active or activated BCRs.

[0024] The solution of these problems by the present invention is based on the surprising discovery that tumor cells in patients with CLL have autonomously active or activated B cell receptors, and that these autonomously active or activated receptors are characterized by the presence of a common epitope that cannot be detected on the corresponding receptors of healthy cells in the same patient. These cells can therefore be specifically recognized and treated with antibodies based on the presence of the autonomously active B cell receptor characterized by the presence of said epitope, without collateralizing healthy B cells that do not possess this property, thereby allowing treatment to be carried out with greater specificity and with fewer undesirable side effects.

[0025] However, during numerous experiments conducted for the present invention, it was surprisingly found that antibodies with particular specificity for these modified receptor regions (epitopes) could not be produced and selected using conventional standard methods. Only after the experimental conditions were adapted to use genetically modified cells in the binding studies and after the modified B cell receptors of these cells were in their native and activated state could suitable antibodies with the desired specificity be obtained. In other words, for the solution proposed by the present invention, it is essential that the cells used in the binding studies for the selection of suitable diagnostic, prophylactic, or therapeutic antibodies present their modified regions (epitopes) largely in their native and activated form. In this regard, so-called pro / pre-B cells were found to be particularly suitable due to their physiological composition. Therefore, the provision of such specific antibodies, and also their functional fragments that also have this specific binding behavior, enables tumor-specific diagnosis and treatments characterized by significantly improved therapeutic success and reduced undesirable systemic effects.

[0026] As mentioned above, biological binding molecules in the form of antibodies or functional fragments thereof, as well as methods for producing (identifying and selecting) such binding molecules that selectively bind to B cells having a B cell receptor with the epitope VL3-21, as well as to denatured epitopes on immunoglobulins of primarily autonomously active membrane-bound B cell neoplasms, are provided. Furthermore, diagnostic and prophylactic as well as therapeutic methods using such binding molecules are proposed, where therapeutic use refers to the inhibition of growth or killing of cells expressing such membrane-bound immunoglobulins. Diagnostic methods refer to the in vitro detection of this receptor subtype, characterized primarily by the presence of VL3-21 on the light chain of the BCR of B cells, particularly in connection with therapeutic decisions to use the proposed antibodies ("companion diagnostics").

[0027] In general, leukemia and lymphoma are attractive targets for treatment with immunotoxins and / or immunocytokines. Responses in patients with B-cell malignancies have been extensively studied in phase I / II clinical trials of immunotoxin activity (Amlot et al., (1993), Blood 82, 2624-2633; Sausville et al., (1995), Blood 85, 3457-3465; Grossbard et al., (1993), Blood 81, 2263-2271; Grossbard et al., (1993) Clin. Oncol. 11, 726-737). While some antitumor responses have been observed, immunotoxin-mediated toxicity to normal tissues has often prevented dose escalation to therapeutic levels. For example, several B cell-specific antigens, such as CD19, CD22, and CD40, have been selected as targets for immunotoxins, which are produced by plant toxins such as ricin A chain and bacterial toxins such as Pseudomonas exotoxin A (PE) (Uckun et al., (1992), Blood 79, 2201-2214; Ghetie et al., (1991), Cancer Res. 51, 5876-5880; Francisco et al., (1995), Cancer Res. 55, 3099-3104).

[0028] Membrane-bound immunoglobulins are well-suited targets for targeted, i.e., specific, immunotherapy. During B-cell development in the bone marrow, each B-cell precursor makes its own, nearly unique, B-cell receptor (BCR) by rearranging individual gene segments.

[0029] Two autonomously active BCR variants (subset 2; subset 4) are known, each differing in its characteristic molecular motif (epitope) (Minici, C. et al., Distinct homotypic B-cell receptor interactions shape the outcome of chronic lymphocytic leukemia, Nature Comm. (2017)). Both variants have different short amino acid sequences specific to each variant. Those skilled in the art will recognize that in addition to the above-listed subsets, other CLL-B cell receptors are also autonomously active. In this regard, the region of subset 2 important for the receptor's autonomously active function is characterized by the amino acid sequences KLTVLRQPKA (SEQ ID NO: 1) and VAPGKTAR (SEQ ID NO: 2) of the light chain, while the region of subset 4 important for the receptor's autonomously active function is defined by the amino acid sequences PTIRRYYYYG (SEQ ID NO: 3) and NHKPSNTKV (SEQ ID NO: 4) of the variable part of the heavy chain. The sequences of subsets 2 and 4 used to generate mouse antibodies within the scope of immunization are shown in SEQ ID NOs: 5 and 6 (vHC;LC) and 7 and 8 (vHC;LC), respectively. For completeness, SEQ ID NO: 17 (VSSASTKG) shows a further target sequence or epitope with specificity for the variable part of the heavy chain of BCRs of subset 4. Thus, in addition to the target sequence (epitope) involved in the formation of the autonomously active state of the BCR (subset 4) according to SEQ ID NOs: 3 and 4, the sequence according to SEQ ID NO: 17 represents a further characteristic property of this subset.

[0030] It should be noted that the discovery and characterization of subsets 2 and 4 as two variants of the B cell receptor in patients with severe disease progression is based on the investigation of a large number of individual case studies and therefore does not imply that the same target sequences (epitopes) that characterize the two known subtypes are not present in the largest possible number of other BCR subtypes and correlate with severe disease progression.

[0031] It should be noted that the present description of the discovery of binding molecules with specificity for BCR subtypes 2 and 4 is understood as preliminary work that led to the discovery of further binding molecules with specificity for epitope VL3-21 according to the present invention, which are themselves the subject of parallel patent applications.

[0032] Although it would be desirable in principle to generate antibodies against both of these subsets by standard methods, e.g., in mice, it was surprisingly observed that immunization with peptides did not result in the formation of the desired specific antibodies. Since immunization with individual chains of the receptor, e.g., the use of the light chain of BCR containing modified sequence regions, also did not achieve the desired success, mice were finally immunized with recombinantly produced soluble forms of BCR (see SEQ ID NOS: 5 and 6). Subsequently, immune cells with the desired specificity could be obtained from these mice and transfected into hybridoma cells by cell fusion. Surprisingly, active antibodies could not be identified by ELISA tests or other standard methods. However, clones identified as potential binding partners in the initial step by ELISA were found after selection to bind nonspecifically or not to the autonomously active receptors (including SEQ ID NOS: 1 and 2) and therefore had to be discarded.

[0033] The methods used leading up to this realization included standard methods such as ELISA and SPR, as well as intracellular expression in fibroblasts with intracellular FACS staining as a binding control.

[0034] After further refinement of the test series, it was found that the screening of suitable binding molecules according to the present invention could not be successfully performed using either free receptors or their fragments, nor membrane-bound or intracellular receptor fragments. Instead, it was observed that the screening was only possible using cell lines in which intact and functional B cell receptors were membrane-bound and displayed in their spheres. In this regard, it is crucial that the BCR with the denatured region (epitope) is present or displayed autonomously and actively in or on these cells. Only with this procedure, whose conditions largely reflect the physiologically native in situ scenario, were it possible to identify antibodies that bind with high specificity and selectivity only to tumor cells, i.e., B cells expressing on their cell membranes BCRs with epitopes characteristic of subset 2 or subset 4 of this cell type, but not to other B cells or their receptors (BCRs) that do not, by definition, represent subset 2 or 4 B cells. In other words, the binding molecule selectively binds to autonomously active or autonomously activated B cell receptors, which are characterized by the presence of a structural domain or epitope (target sequence) and which is responsible for the autonomously active or activated state of the B cell receptor.

[0035] Furthermore, the use of pro / pre-B-arrested cells obtained from "triple knockout" mice (TKO), despite their difficult handling and laborious acquisition, has proven well suited to express these receptors and to be used to identify them in a range of test systems. The pro / pre-B cell stage is naturally designed to carry out BCR maturation and sorting, and this cell stage is particularly well-suited to correctly fold even "challenging" BCR components based on their enzymatic properties (e.g., chaperones) and present them on their surface in a fully physiologically native form. The deletions (knockouts) described below prevent the desired BCR alterations from occurring through recombination or the use of a "surrogate light chain." The use of these cells or this cell type of pro / pre-B arrested cells for the expression and presentation of BCRs within the scope of the selection of antibodies with a selective-specific binding behavior to autonomously active B cell receptors or activated B cell receptors provides a selection platform characterized by a much higher quality compared to the systems conventionally used for prior art selection, which justifies the high expenditure of the use of primary TKO cells and their culture over several passages, respectively.

[0036] After selecting suitable hybridoma cells as described above, it is possible to obtain a large amount of antibodies suitable for diagnostic, preventive and / or therapeutic purposes in the form of monoclonal antibodies. The binding sites of the antibodies can be confirmed by sequencing the DNA of these cells (see SEQ ID NOs: 9 and 10). Corresponding methods are known to those skilled in the art and are commercially available. In this regard, it is advantageous to obtain a larger number of hybridoma cells and select the cells with the best binding activity (specificity and binding strength / affinity).

[0037] Using the genetic information for the binding site thus obtained, the encoding sequence was inserted into an expression plasmid together with the DNA of the human antibody sequence, and humanized monoclonal antibodies with the desired specificity were produced using conventional recombinant methods. Based on their unique specificity, these humanized antibodies demonstrated superior diagnostic specificity or prophylactic and therapeutic effects compared to conventional diagnostic tools and agents, with relatively few side effects. Those skilled in the art will understand that these humanized antibodies can be mass-produced using biotechnology techniques. Purification of synthesized antibodies can be performed using standardized methods, such as a combination of precipitation, filtration, and chromatography, which are well known to those skilled in the art. It is important to note that the antibody must not be denatured and that possible foreign substances, such as proteins, pyrogens, and toxins, must be quantitatively removed.

[0038] The desired antibody is preferably expressed in a system in which the antibody is glycosylated, e.g., particularly human glycosylation. Such systems are well known to those skilled in the art and include the use of insect cells (S2 cells), mammalian cells (CHO cells), and particularly preferably, human cells such as HEK293T cells.

[0039] A well-purified antibody may itself be therapeutic if it has an isotype that elicits a specific immune response, such as an IgG subtype that mediates an immune response against tumors via Fc receptors.

[0040] On the other hand, antibodies can also exist as fragments. In this case, it is important that the antigen-binding site is present in the fragment, i.e., that it is a functional fragment. Such fragments can be generated, for example, as F(ab) fragments by protease treatment. Since these fragments are truncated in the antibody constant region, it is advantageous and therefore preferable to insert an effector molecule for killing neoplasms into them.

[0041] In an alternative preferred embodiment, the antibody is provided with a conjugate to enhance its effectiveness. The conjugate is a neoplasm-killing moiety that can kill such neoplasms as a direct or indirect effect. One example of such a conjugate is the attachment of ricin to the antibody, where the preferential covalent attachment is achieved, for example, using a chemical crosslinker. Such molecules and methods are extensively described in Chapter 11, "Immunotoxin Conjugation Techniques," of "Bioconjugate Techniques" by Greg T. Hermanson.

[0042] According to a further preferred embodiment, antibodies can also be present in modified form as biological binding molecules, for example in the form of fusion proteins with T cell-specific activation domains. To generate these so-called chimeric antigen receptors (CARs), T cells must first be collected from the patient's peripheral blood and genetically modified in vitro to express the CAR on their cell surface. These modified T cells can then be reintroduced into the patient, enabling CAR T cell immunotherapy (see, for example, N Engl J Med. 2014 Oct 16; 371 (16): 1507-17. doi: 10.1056 / NEJMoal407222).

[0043] For therapeutic use, the antibodies are preferably used in a composition that includes a pharmaceutically acceptable carrier.

[0044] Pharmaceutically acceptable carriers are those that are physiologically acceptable to the patient receiving treatment and maintain the therapeutic properties of the compound being administered.An exemplary pharmaceutically acceptable carrier is physiological saline.Other suitable physiologically acceptable carriers and their formulations are known to those skilled in the art and are described, for example, in Remington's Pharmaceutical Sciences (18. Ausgabe), Hrsg. A. Gennaro, 1990, Mack Publishing Company, Easton, PA.

[0045] A further possibility of therapeutic application of the binding molecules according to the invention is the apheresis method known per se, in which the treatment of a patient's blood or a blood sample takes place outside the patient's body in the sense of "blood washing".

[0046] For example, the antibodies according to the invention can be used in an apheresis system for isolating leukemic cells from a patient's blood sample, for which in principle various methods are suitable, as known to those skilled in the art.

[0047] According to a first exemplary embodiment, the antibodies may be conjugated to magnetizable particles (beads) (e.g., Dynabeads). The blood is then contacted with the particles outside the patient's body after administration of an anticoagulant. Ideally, at least one particle per tumor cell, preferably 10-100 particles, is used for this purpose. Here, particles with a size of, for example, less than 20 μm typically contain multiple antibodies with the same specificity (the number of particles exceeds 5,000 per μl of blood). These particles are then bound together using a magnet, and the remaining purified blood can be returned to the patient. This treatment significantly reduces the number of tumor cells in the patient's blood. According to another embodiment, the particles are larger than 20 μm in size and contain a high number of antibodies per particle (>100, >1,000). Therefore, a single particle can bind and remove many lymphocytes (tumor cells). These particle-cell conjugates are then removed by classical centrifugation, as is commonly used in apheresis. The time required varies depending on the type of particle and device and must be determined experimentally.

[0048] In a further embodiment, both particle-cell conjugates (especially when large particles with a diameter of more than 20 μm are used) and free particles not bound to cells can be separated from blood using fine networks. Such networks are commercially available, for example, as so-called "cell strainers." Methods for conjugating antibodies to particles are well known to those skilled in the art. Instructions are provided, for example, by Dynal to its customers.

[0049] For diagnostic purposes, it is preferred to use antibodies in standardized methods such as flow cytometry or immunohistochemistry. Advantageously, the antibodies, biological binding molecules or functional fragments thereof proposed for diagnostic purposes have a murine backbone. Detection in a flow cytometer is advantageously carried out using a secondary antibody, or alternatively, preferably using a fluorescent dye directly bound to the antibody, the biological binding molecule or functional fragment thereof.

[0050] For stable storage, it may be advantageous and therefore preferred to provide the antibody or fragment thereof in a stabilized form. For this purpose, drying may be carried out, for example, in a stabilized salt buffer. Such a buffer may be, for example, phosphate-buffered saline (PBS), which is known to those skilled in the art. Suitable forms of drying include, for example, lyophilization or freeze-drying.

[0051] Individual aspects of the invention are explained in more detail below by way of examples.

[0052] Before proceeding to a detailed description of the experimental procedures, please refer to the following explanations.

[0053] The production and identification of antibodies that selectively bind to modified B cell receptors was characterized by a major unexpected problem. Hybridoma generation was performed using standard methods. Supernatants from hybridoma groups were pooled and examined for positive binding events using ELISA (soluble B cell receptor on ELISA plates). The positive pool was separated, and individual clones were tested. Surprisingly, no positive clones were identified by ELISA. The positive ELISA signal of the pool was subsequently determined to be nonspecific binding.

[0054] To generate a better epitope for antibody recognition, the BCR light chain was expressed in fibroblasts, which ensured correct folding of the protein carrying the motif (epitope) involved in autonomous signaling. Intracellular FACS analysis was performed using these cells. No positive clones (antibodies) could be identified.

[0055] For this reason, RAMOS cells (a human Burkitt's lymphoma cell line) were modified in a separate experiment, and they displayed a functionally modified BCR. This ensured complete correct biogenesis, folding, and modification of the BCR. To achieve this, the cells' own BCR was deleted using CRISPR, and then the "CLL receptor" was molecularly reconstituted (electroporation of a CMV vector). These cells were then used to test for positive binding events. Again, no positive clones were detected using FACS.

[0056] Surprisingly, in contrast, the use of mouse TKO ("triple knockout") cells (pro / pre-B arrested cells) into which the CLL receptor was introduced by gene shuttle yielded positive clones, despite the fact that human cell lines could not guarantee this. These cells characteristically have three knockouts in their genome: - Knockout of RAG2 prevents somatic recombination of the intrinsic heavy and light chains of immunoglobulins, thus eliminating the endogenous formation of BCRs. This leads to the arrest, blocking or "freezing" of correspondingly processed B cells at this stage of development. Since RAG1 and RAG2 are known to initially form a complex that allows normal VDJ rearrangement, knockout of RAG1 is a means of achieving the same effect and is therefore an alternative to knockout of RAG2, and is within the teachings of the present invention.

[0057] - Deleting Lambda5, which is part of the surrogate light chain, prevents the formation of pre-BCRs. Because pre-BCRs are autonomously active, this interferes with the detection of autonomously active receptors. Now, as new BCRs are cloned into cells, the pre-BCRs are undesirable because they will appear on the surface with the desirable heavy chain (HC) in combination with the undesirable surrogate light chain, preventing sorting.

[0058] - Knockout of SLP65, the most important adaptor protein in the BCR signaling pathway, prevents the activation of TKO cells by rearranged BCRs in some cases.

[0059] Combination knockout of RAG2 or RAG1 and Lambda5 blocks the transition from the pro-B to pre-B cell stage, classically characterized by early rearrangement of the VDJ segment of the heavy chain (HC). Hence, they are pro / pro B cells.

[0060] Knockout of RAG2 or RAG1 and Lambda5 is sufficient for BCR expression and selection of appropriate antibodies, and BCR activity can be measured by reconstitution with inducible SLP65.

[0061] The screening method used here involves measuring Ca flux after induction of SLP65 using FACS analysis and Ca receptors such as Indo-1. 2+ These methods are known to those skilled in the art (see M. Duehren-von Minden et al.; Nature 2012).

[0062] The first two knockouts ensure that only the "BCR of interest" is expressed on the surface. By reconstituting cells with inducible SLP65, we can further characterize the function of the expressed BCR and thus verify the autonomously active state of the surface BCR before sorting.

[0063] BCR expression was measured by FACS using anti-IgM and anti-LC antibodies. For this purpose, some cells were harvested and stained with 5 μl of each antibody in a total volume of 100 μl in PBS.

[0064] Using these cells as "targets," we successfully used FACS to identify antibodies that specifically bind to the altered region that underlies and characterizes autonomous BCR activation, even though they were unable to bind to the same receptor type on RAMOS cells.

[0065] For this purpose, cells bearing the "BCR of interest" on their surface were first incubated with the pooled supernatants. After several washing steps, bound antibodies were detected using a secondary antibody. For specific selection, TKO cells (TKO) expressing different versions of the "BCR of interest" were used. The sorting matrix shown in Figure 1 is a typical example of the selection of CLL subset 2BCR and was used to identify and select positive clones. To facilitate identification, hybridoma supernatants were pooled and assayed. Groups that showed binding were separated, and the binding of each hybridoma supernatant was tested.

[0066] Confirmation that the selected antibodies specifically bind to the modified BCR and not other BCR variants was performed using two blank samples: cells without a BCR (see Figure 1A) and cells with a non-CLL BCR (see Figure 1E). Primary B cells from the blood of leukemia patients were examined for binding using FACS. The selected antibodies were able to specifically identify the BCR that exhibited the target structure. This was confirmed at the genetic level. Samples without this target structure did not exhibit binding.

[0067] In this study, all autonomously active cells of CLL subset type 2 used were found to have a specific mutation in the V region of the BCR light chain in addition to a specific mutation (R110G). This epitope variant was identified as VL3-21 (SEQ ID NO: 18) and constitutes the subject of the present invention. This epitope has been shown to be present on the BCR of tumor cells in approximately 30% of all CLL cases. However, this epitope is also present on healthy cells. Therefore, it is a tumor-associated epitope that can be used for tumor diagnosis and treatment. Notably, due to the large number of variants in this V region, the variant VL3-21 described herein is expressed by less than 5% of "healthy," i.e., tumor-free, B cells. This offers therapeutic potential for preferentially eliminating neoplastic B cells (tumor cells) and causing much less damage to healthy, non-tumorous B cells compared to the conventional use of nonspecific antibodies against the BCR. Therefore, the use of the proposed antibody or functional fragment thereof specific for VL3-21 according to the present invention allows for treatment with much higher specificity and less burden on the patient. Furthermore, antibodies against VL3-21 can be used for so-called companion diagnostics, for example, in CLL patients. In this diagnostic, the presence of the target structure VL3-21 of a therapeutic antibody on tumor cells is detected, proving the likelihood of success of the corresponding treatment.

[0068] The invention will be explained in more detail below by way of example in view of the drawings, in which: [Brief explanation of the drawings]

[0069] [Figure 1] FIG. 1 shows a selection matrix representative of the selection of CLL subset 2BCR. [Figure 2] FIG. 1 shows an example of analysis using an antibody specific to subset 2.

[0070] Example 1 The starting point for the production of triple knockout cells (TKO) is the creation of transgenic mice with knockouts of the genes Lambda5, RAG2, and SLP65 (Duehren von Minden et al., 2012, Nature 489, pp. 309-313). The creation of such mice is known to those skilled in the art and is included in the prior art. To obtain the cells, the bone marrow of the femurs was extracted after sacrificing the mice. The cells thus obtained were then cultured under conditions that promote the survival of pro / pre-B cells (37°C, 7.5% CO2, Iscoves medium, 10% FCS, P / S, mouse IL-7). After several passages, FACS sorting was performed for control purposes, and pro / pre-B cells were then sorted and returned to culture. The markers used for this purpose are known to those skilled in the art.

[0071] To reconstitute the "target BCR," the corresponding sequences encoding the heavy chain (HC) and light chain (LC) were synthesized and then cloned into their respective expression vectors with a CMV promoter. These were then introduced into a packaging cell line (Phoenix cell line) by lipofection. After 36 hours of incubation, the viral supernatant was removed and used for spinfection of TKO cells. Both the procedures for obtaining the supernatant and spinfection of TKO cells are well known and familiar to those skilled in the art.

[0072] The structural characteristics of the subset 2 B cell receptor were taken from the corresponding literature (see above). Exemplary CLL subset 2 VH and complete LC DNA segments were synthesized by a contract manufacturer using standard methods. These were then fused to mouse IgG1 constant segments using PCR and cloned into a CMV vector. The sequence of the completed vector was confirmed by Sanger sequencing.

[0073] CLL subset 2 VH (SEQ ID NO: 5): [ka]

[0074] CLL subset 2LC (SEQ ID NO: 6): [ka]

[0075] A human cell expression system based on HEK293T cells was used to express CLL subset 2 IgG1. A polyethyleneimine (PEI)-based protocol was used for transfection. After several passages, the supernatants were pooled and the media containing the bound cell supernatant was purified using a protein G column. The purity and quality of the soluble subset 2 IgG1 were determined by Western blotting.

[0076] Monoclonal antibodies were produced in mice using standard methods, followed by hybridoma cell generation. Screening for positive clones was not performed using conventional ELISA. Because the target structure is a membrane-bound receptor, it was paramount to demonstrate potential antibody binding in a cell line, i.e., while largely preserving the cell physiological state inherent to this cell type. First, FACS analysis was used to examine binding events in a group of pooled supernatants. To this end, various CLL subset 2 BCR variants were expressed on the surface of a cell line (TKO) that cannot express the BCR itself. This allowed us to initially identify supernatants that showed antibody binding. Subsequently, supernatants from individual hybridoma clones were examined in more detail for their binding, identifying highly specific clones with high affinity.

[0077] In the screening method, the following combinations of heavy chain (HC) and light chain (LC) of the corresponding CLL-BCR were used in the preceding transfection using different vectors, and these combinations were used on the surface of the BCR-reconstituted system: Control (transfection vector without BCR) (see Figure 1A) A vector (VL3-21) carrying HC / LC typical of CLL subset 2 (see Figure 1B) A vector with a LC typical of non-CLL subset 2HC / CLL subset 2 (VL3-21; lacking the target motif R110G) (see Figure 1C) Vectors with HC / non-CLL subset 2 LC typical of CLL subset 2 (see Figure 1D) Vector with one non-CLL subset 2HC / one non-CLL subset 2LC (see Figure 1E) A vector with a HC / LC typical of CLL subset 2 (VL3-21; containing the mutation R110G (target motif)) (see Figure 1F).

[0078] This selection procedure is illustrated schematically in Figure 1 using the example of the CLL subset 2BCR, where the term "TKO" refers to TKO cells (see above).

[0079] In the first sorting round, supernatants from several clones were combined and examined for their binding profile to the sorting matrix. A positive binding profile was provided if specific binding to the "BCR of interest" was demonstrated. Groups showing such profiles were isolated, and the binding profiles of individual clones were recharacterized by the sorting matrix within the second sorting round. Monoclonal antibody binding was verified using a FACS binding assay with a fluorescently labeled anti-mouse IgG antibody. Designations include: A) no BCR (control); B) BCR typical of CLL subset 2; C) BCR with random heavy chains and CLL subset 2-typical light chains; D) BCR with CLL subset 2-typical heavy chains and random light chains; E) BCR with random heavy and light chains (control; non-CLL subset 2-typical BCR); F) CLL subset 2-typical BCR with a mutation in the target motif (R110G) (control). It should be noted that the VL3-21 variant is used as the light chain in cases B, C, and F in Figure 1.

[0080] Based on the finding that the antibody binds only to cells bearing the target structure (CLL subset 2BCR; Figure 1B), we can conclude that an antibody is present here that specifically binds to cells bearing an autonomously active receptor.

[0081] In this regard, we found that the correct expression of the BCR required for detection requires the use of cells at the stage of pro / pre-B cell development. These cells are genetically primed to express the new BCR by correct folding and expression on their surface. Inactivation (knockout) of RAG2 and Lambda5 prevents the expression of endogenous BCRs or pre-BCRs. Deletion of SLP65 followed by inducible reconstitution of SLP65 allows the characterization of the activity level of the "BCR of interest."

[0082] To determine the amino acid sequences of the selected monoclonal antibodies, mRNA was isolated from individual hybridoma clones, and cDNA was generated from them and amplified using anchored PCR (Rapid expression cloning of human immunoglobulin Fab fragments for the analysis of antigen specificity of B cell lymphomas and anti-idiotype lymphoma vaccination; Osterroth F, Alkan O, Mackensen A, Lindemann A, Fisch P, Skerra A, Veelken H., J Immunol Methods 1999 Oct 29;229(1-2):141-53).

[0083] After identifying and sequencing the CDRs (critical for binding), they were transferred to a human antibody framework by PCR. For this purpose, VH sequences were generated in silico from the human FR and mouse CDR regions and then synthesized as DNA fragments. These were then fused to human IgG1 by PCR and cloned into a vector suitable for expression.

[0084] In addition to intact immunoglobulins, synthetic peptides displaying regions of autonomous signaling competence were also used to generate monoclonal antibodies.

[0085] The specific monoclonal antibody for subset 2 has been sequenced and is the subject of a separate patent application.

[0086] Upon sequencing, the following amino acid sequences were determined, SEQ ID NO: 9 corresponds to the variable part of the heavy chain (HC), SEQ ID NO: 10 corresponds to the variable part of the light chain (LC), and the marked regions represent CDR1, 2 and 3 in the order shown.

[0087] SEQ ID NO: 9 (AVA-mAb01 HC) [ka]

[0088] SEQ ID NO: 10 (AVA-mAb01 LC) [ka]

[0089] The partial sequences of the heavy chain corresponding to CDR1, CDR2 and CDR3 according to SEQ ID NO: 9 are shown in SEQ ID NOs: 11 to 13, and the partial sequences of the light chain corresponding to CDR1, CDR2 and CDR3 according to SEQ ID NO: 10 are shown in SEQ ID NOs: 14 to 16.

[0090] SEQ ID NO: 11 (AVA-mAB01 CDR1 HC) GFSLTSYG SEQ ID NO: 12 (AVA mAB01 CDR2 HC) IWRGGGT SEQ ID NO: 13 (AVA mAB01 CDR3 HC) ARSRYDEEESMNY SEQ ID NO: 14 (AVA mAB01 CDR1 LC) GNIHSY SEQ ID NO: 15 (AVA mAB01 CDR2 LC) NAKT SEQ ID NO: 16 (AVA mAB01 CDR3 LC) QHFWNTPPT

[0091] The above procedure is an example for generating antibodies specific for CLL subset 2. The same process was performed using subset 4 specific sequences and isotypes.

[0092] The representative CLL subset 4 VH and complete LC DNA segments were synthesized by a contract manufacturer using standard methods.Then, they were fused with mouse IgG1 constant segments using PCR and cloned into CMV vectors.The sequence of the completed vector was confirmed by Sanger sequencing.

[0093] CLL subset 4HC (SEQ ID NO: 7): [ka]

[0094] The bolded region indicates the target sequence (epitope) of the variable part of the heavy chain of the subset 4 BCR that is responsible for its autonomously active state (see SEQ ID NOs: 3 and 4).

[0095] CLL subset 4LC (SEQ ID NO: 8): [ka]

[0096] Subsequent studies with discarded hybridoma cell lines led to the surprising discovery of binding molecules according to the invention specific for VL3-21 of the light chain of the B cell receptor. The supernatants of each of these hybridoma cell lines showed binding to the TKO cell lines shown in Figure 1B, Figure 1C, and Figure 1F.

[0097] The sequence of the thus identified B cell receptor-specific monoclonal antibody bearing the epitope VL3-21 was determined, yielding the following amino acid sequence, in which SEQ ID NO: 25 corresponds to the variable portion of the heavy chain (HC), SEQ ID NO: 26 corresponds to the variable portion of the light chain (LC), and the marked regions represent CDR1, 2, and 3 in the order shown.

[0098] SEQ ID NO: 25 (AVA-mAb02 HC) [ka]

[0099] SEQ ID NO: 26 (AVA-mAb02 LC) [ka]

[0100] The partial sequences of the heavy chain corresponding to CDR1, CDR2 and CDR3 according to SEQ ID NO: 25 are shown in SEQ ID NOs: 19 to 21, and the partial sequences of the light chain corresponding to CDR1, CDR2 and CDR3 according to SEQ ID NO: 26 are shown in SEQ ID NOs: 22 to 24.

[0101] SEQ ID NO: 19 (AVA-mAB02 CDR1 HC) GYTFTDYA SEQ ID NO: 20 (AVA-mAB02 CDR2 HC) ISTYYGDS SEQ ID NO: 21 (AVA-mAB02 CDR3 HC) SRDTSNFDY SEQ ID NO: 22 (AVA-mAB02 CDR1 LC) QDINSH SEQ ID NO: 23 (AVA-mAB02 CDR2 LC) RANR SEQ ID NO: 24 (AVA-mAB02 CDR3 LC) LQYDEFPRT

[0102] Example 2 Using the teachings of the present invention, a small amount of peripheral blood was collected from a patient. For analysis, 100 μl of blood was transferred to a reaction vessel and 2 ml of PBS-BSA buffer was added. The sample was then centrifuged at 1500 rpm for 5 minutes in an Eppendorf 5804 centrifuge. The supernatant was discarded, and the pellet was mixed thoroughly. Antibodies were then added. Staining for the following surface parameters was performed before incubating for 15 minutes at room temperature in the dark: 1) CD19-FITC, 2) CD5-PE, and 3) antibodies specific for VL3-21 (APC). Lysis was then initiated to lyse red blood cells. As previously described, cells were washed twice with PBS-BSA buffer and resuspended in 500 μl of 0.1% PBS-BSA buffer. Cells were stored in the dark at 2-8°C until analysis on a flow cytometer.

[0103] FACS analysis was performed on a BDCalibur. The individual laser and detection parameter settings were performed according to the device manufacturer's instructions and are well known to those skilled in the art. The raw data from the analysis were then evaluated using FlowJo analysis software. Lymphocyte populations were first selected and labeled in an FSC / SSC blot. For this selection, CD19-positive B cells were then focused and analyzed for binding of a VL3-21-specific antibody. Figure 2 shows an example of such an analysis using an antibody specific for VL3-21. In a first step, CD19-positive B cells were selected for further analysis (left panel). These were then examined for binding of specific antibodies (right panel).

[0104] Example 3 The antibody according to the present invention, which has specificity for the BCR with VL3-21, was used in an apheresis system to separate leukemia cells from a patient's blood sample.

[0105] Peripheral blood was collected from one patient (EDTA blood collected from a blood collection tube). Lymphocyte count was measured using a cell counter and found to be 80,000 lymphocytes / μl. To determine the "tumor burden," i.e., the burden of tumor cell material in the sample, immunophenotyping was performed by FACS (using the CLL standard panel WHO Tumorload). Cells bearing CLL epitopes were then stained with the antibody of the present invention to detect cells positive for this epitope. 500 μl of this blood was then mixed with 5 × 10 MACS MicroBeads (Miltenyi Biotech) conjugated with a specific antibody. After shaking the mixture at room temperature for 5 minutes, the blood was applied to a Miltenyi LS column. The particle-conjugated lymphocytes remained in the column, while the lymphocyte-free blood was collected as flow-through. After double column purification (according to the manufacturer's instructions), lymphocyte counts of less than 5000 lymphocytes / µl could be determined by FACS. In control experiments using blood from CLL patients lacking the VL3-21 BCR on their leukemia cells, this purification did not significantly deplete the blood B cells, as expected.

Claims

1. A composition for the diagnosis, prevention and / or treatment of a cancer disease, the composition comprising a biological binding molecule which is an antibody or a fragment thereof having an antigen-binding site, the antibody selectively binding to a B cell receptor characterized by the presence of a light chain having the sequence according to SEQ ID NO: 18, and which comprises CDR1, CDR2 and CDR3 of a heavy chain variable region represented by SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21, respectively, and CDR1, CDR2 and CDR3 of a light chain variable region represented by SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24, respectively, and the amino acid sequences of the heavy and light chain variable regions are represented by SEQ ID NO: 25 and SEQ ID NO: 26, respectively.

2. The composition according to claim 1 for the diagnosis, prevention and / or treatment of malignant B-cell neoplasms.

3. The composition according to claim 1 or 2, which is used in apheresis or CAR-T cell immunotherapy.

4. 3. The composition according to claim 1 or 2, used in diagnostics for the detection of B cells having a B cell receptor characterized by the presence of a light chain according to SEQ ID NO: 18.

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