MXPA00010245A - CD19xCD3 SPECIFIC POLYPEPTIDES AND USES THEREOF - Google Patents

Abstract

Described are novel single-chain multifunctional polypeptides comprising at least two binding sites specific for the CD19 and CD3 antigen, respectively. Further provided are polypeptides, wherein the above-described polypeptide comprises at least one further domain, preferably of pre-determined function. Furthermore, polynucleotides encoding said polypeptides as well as to vectors comprising said polynucleotides and host cells transformed therewith and their use in the production of said polypeptides are described, In addition, compositions, preferably pharmaceutical and diagnostic compositions are provided comprising any of the afore-described polypeptides, polynucleotides or vectors. Described is also the use of the afore-mentioned polypeptides, polynucleotides and vectors for the preparation of pharmaceutical compositions for immunotherapy, preferably against B-cell malignancies such as non-Hodgkin lymphoma.

Description

INNOVATIVE POLYPEPTIDES CD19 X CD3 SPECIFIC AND USED The present invention relates to single-stranded multifunctional polypeptides: innovators comprising at least two specific binding sites for the CD19 and CD3 antigens, respectively. The present invention furthermore relates to a polypeptide, wherein the polypeptide described above comprises at least one additional domain, preferably of predetermined function. In addition, the present invention relates to polynucleotides encoding these polypeptides as well as the vectors comprising said polynucleotides and host cells transformed therein as well as their use in the production of the polypeptides. Additionally, the present invention is . It is used with compositions, preferably pharmaceutical and diagnostic compositions, comprising any of the polypeptides, polynucleotides or vectors described above. A further objective of the present invention is the use of the aforementioned polypeptides, polynucleotides and vectors for the preparation of the pharmaceutical compositions for immunotherapy, preferably against B cell malignancies. ly lymphoma that is not Hodgkin type. Several documents are cited through the text of this specification. Each of the documents cited herein (including any specification, instruction, etc. of the manufacturer) is incorporated by reference; however, there is no admission that any of the cited documents are in fact the prior technology of the present invention. Despite the medical importance, research on B-cell-mediated diseases such as non-Hodgkin lymphoma has produced only a number of clinically usable data and conventional approaches to cure these diseases remain tedious and unpleasant and / or have a high risk of relapse. For example, although a high dose of chemotherapy as a first treatment for a high-grade lymphoma that is not of the Hodgkin type can improve overall survival, about 50% of patients still die from this disease (2-4). Furthermore, chronic lymphatic leukemia similar to non-low-grade Hodgkin lymphoma and mantle cell lymphoma are still incurable. This has stimulated the search for alternative strategies such as immunotherapy. Antibodies directed against cell surface molecules are defined as CD antigens that represent a unique opportunity for the development of therapeutic reagents. The expression of certain CD antigens is highly restricted to lymphohematopoietic cells of specific lineage and for several previous years, targeted antibodies and specific lymphoid antigens have been used to develop treatments that were effective in the vi or in animal models (5- 13). On this respect CD19 has proven to be a very useful target. CD19 is expressed in the entire B lineage from the pro cell B to the mature B cell, is not expressed, is expressed uniformly in all lymphoma cells, and lacks the stem cells. An interesting modality of this application of a bispecific antibody with a specification for CD19 and the other for the CD3 antigen in T cells. However, this is how the hardly available bispecific antibodies suffer from a low cytotoxicity by the T cells and the need for costumer agents in order to exhibit satisfactory biological activity.

In this way, the technical problem highlighted by the present invention is that of providing elements and methods useful for the treatment of diseases mediated by B cells as well as different forms of lymphoma that is not of the Hodgkin type. The solution to this technical problem is achieved by providing the embodiments characterized in the claims. Correspondingly, the present invention relates to a single single chain multifunctional polypeptide comprising (a) a first domain comprising a binder site of an immunoglobulin chain or an antibody that specifically recognizes the antigen I '. C.D19; and (b) a second domain comprising an agglomerating site of an immunoglobulin chain or an antibody that specifically recognizes the CD3 antigen. The terms "first domain" and "second domain" according to the present invention represent that a binder site is directed against the B-cell maker CD19 plate, which spreads uniformly over a vast majority of the 5 malignant B cells. , the other binder site is directed against the human T cell antigen CD3. The term "binding site" which is used in the present invention denotes a domain that undertakes a three-dimensional structure capable of agglomerating specifically to an epitope similar to native antibodies, free fragments of scFv or one of its corresponding immunoglobulin chains, respectively VH string. Thus, this domain can compromise the VH and / or VL domain of an antibody or an immunoglobulin chain, preferably at least the VH domain. On the other hand, these binding sites contained in the polypeptide of the invention can therefore comprise at least one complement determining region (CDR) of an antibody or an immunoglobulin chain that recognizes the CD19 and CD3 antigens, respectively. In this regard, it has been noted that the domains of the binding sites present in the polypeptide of the invention may not be derived from other antibodies but also derive from other CD19 or CD3 binding proteins, such as receptors or ligations with a surface that occurs naturally. According to the invention, this binder site is comprised in a domain. The term "multifunctional polypeptide" is used herein denotes a polypeptide comprising at least two amino acid sequences '-rivadas of different origins, that is, of two different molecules, optionally derived from two different species where at least two of the origins specify the agglomerating sites.

Correspondingly, binder sites specify functions or at least some -.licons of the multifunctional peptide. These l (polypeptides include, for example, single-stranded bispecific antibodies (bsc) The term "single chain" used according to the present invention means that the first domain and the second domain of the polypeptide are covalently linked, preferably in the form of a colinear amino acid sequence encoded by a nucleic acid molecule. CD19 denotes an antigen that is expressed in lineage B as in pro cell B and the mother B cell, spills, is expressed uniformly in all cells? ... lymphoma and lacks stem cells (8, 14). CD3 denotes an antigen that is expressed in T cells as part of a multimolecular T cell receptor complex and that consists of three chains ? r different from CD3e, CD3d and CD3 ?. Clustering of CD3 in T cells, for example, through immobilized antibodies •••• t-CD3, leads to T cell activation similar to T-cell receptor coupling but independent of its typical one hundred specificity. Actually, most of the antibodies "~" iti-CD3 recognize the CD3e chain. Antibodies that specifically recognize < The antigen CD19 or the CD3 antigen are described in the prior art, for example in (24), (25) and (43), respectively and can be generated by conventional methods known in the art. Bispecific CD19xCD3 antibodies that do not fill a single-stranded format, upon re-targeting the T-cell cytotoxicity in lymphoma cells in an independent manner.

MHC have been shown to be effective in vi tro (5, 6, 9-? '., 13, 43), in animal models (7, 28) as well as in pilot clinical studies (12, 29, 30). Up to this point these antibodies were constructed through hybrid-hybridoma techniques, by covalent attachment of monoclonal antibodies (31) or by a diacorporal approach (43). The most clinical studies ..they have been hampered by the fact that these ..? t antibodies have a low biological activity in such a way that high doses have been applied and that ilication of antibodies alone did not provide a beneficial therapeutic effect. In addition, the availability of clinical grade material was limited. Without adhering to a particular theory, it is believed that by using the format similar to the bispecific antibody as defined above, they are generated : this forms the polypeptides as the CD19xCD3 specific antibodies that usually have the ability to destroy CD19-positive target cells by recruiting cytotoxic T lymphocytes without any need for pre-stimulation or co-stimulation of the T cell. This is an acute contrast to all bispecific CD19xCD3 molecules produced according to other molecular formats and usually does not depend on the particular specificities of the CD19 or CD3 antibodies used to construct, for example, the bispecific single chain antibody.

The independence of the pre-stimulation and / or co-stimulation of the T cell can contribute substantially to the exceptionally high cytotoxicity mediated by the polypeptide of the invention as exemplified by the bispecific antibody CD19xCD3 described in the examples. An additional advantageous property of the li-peptide of the invention is that due to its small, relatively compact structure it is easy to .produce and purify, thus avoiding the problems of < LS productions, the occurrence of by-products defined as diseased, or laborious purification procedures (15-19) for antibodies Specific CD19xCD3 hitherto produced from hybrid hybpdoma, through chemical bonding or by renaturation from bacterial inclusion bodies. Next, the advantageous and unexpected properties of the polypeptide of the invention will be discussed in an unlimited manner guided by the appended examples, including some of the forms of preference loyalty of the invention referred to in this document, which illustrate a broad concept of the invention. present invention. In accordance with the present invention, a eukaryotic expression system was used that has been developed for the production of recombinant single-chain bispecific antibodies (1) in order to generate a recombinant CD19xCD3 bispecific antibody through expression in CHO cells . The fully functional antibody from the superfluous culture was easily purified through its C-terminal histidine tag on a Ni-NTA chromatography column. The specific agglomeration of CD19 and CD3 was demonstrated through FACS analysis. The resulting molecule bscCD19xCD3 (bispecific single chain CD19xCD3) of the invention showed some unexpected properties: it induced a high cytotoxicity of the lymphoma (r? G? C-? To the T cell viin and in vi.V even at very low concentrations of 10-100 pg / ml and low E (effector): T (white) in ratios of 5: 1 and 2.5: 1 significant specific lysis of lymphoma cell lines was observed, in addition, 3μg to 10μg The bscCD19xCD3 molecule of the invention in compassionate use showed a clear and significant improvement of the medical condition compared to the published CD19xCD3 antibodies hitherto published through the hybrid-hybridoma techniques or through diabody approaches (which also present a different format) which show a cytotoxic activity in the range of several nanograms / ml or up to μg / ml, the bscCD19xCD3 antibody of the invention seems to be much more effective (5-7, 27, 43) as documented, for example In the examples of 1, G Appendices 4, 5 and 7. Even the low concentrations of bscCD19xCD3 of the invention had the ability to induce a rapid cytotoxicity of targeted lymphoma (after 4 h) with low E: T ratios. without the need for any pre-stimulation of the T cell. In contrast, a conventional CD19xCD3 bispecific antibody (5-7, 27) did not show significant cytotoxic activity under these conditions (especially without pre-stimulation of the T cell, with a low ratio of E: T) even at high concentrations up to 3000 ng / ml. Although the induction of cytotoxic activity without pre-stimulation has also been reported in the case of another conventional CD19xCD3 antibody, this effect has been achieved only at high concentrations and high E: T ratios (lOOng / ml, 27: 1) ( 9) compared to the bscCD19xCD3 of the invention (100 pg / ml 2.5: 1). Moreover, a cytotoxic effect of this antibody has been observed only after 1 day of pre-stimulation with the bispecific antibody, while the bscCD19xCD3 of the invention had already induced cytotoxicity directed to the lymphoma after 4 hours. To the knowledge of the inventors, this rapid and specific cytotoxic activity of the unstimulated cells at these low concentrations and ratios of E: T have not been described for other bispecific antibodies used up to the time. Although recently an anti-pi05HER2 / anti-CD3 biospecific antibody F (ab) 2 has been shown to induce a cytotoxic activity at similar concentrations as those of bscCD19xCD3 of the invention, this antibody required 24 hours of pre-stimulation with TL-2 (32) . This is how the antibody bscCD19xCD3 of L ivención reveals unique cytotoxic properties that discriminate this molecule from other bispecific antibodies that have been described. The bscCD19xCD3 of the invention mediates the cytotoxic erecti which are specific antigens demonstrated by the facts that: this antibody did not produce lysis of the NCI and L363 plasmacytoma cell lines which are cell lines of the B lineage that do not express the antigen CD19; and - that the cytotoxicity against the lymphoma cells can be blocked by the antibody, parental nti-CD19 HD37. (The HD37 antibody is derived from hybridoma HD37 (22)). By blocking the perforin pathway through calcium deficiency with completely blocked EGTA, it is inferred that the mediated cytotoxicity of bscCD19xCD3 suggests that the specific lysis is a mediated effect of the T cell instead of a direct effect of the antibody. Taken together, the bscCD19xCD3 antibody constructed in accordance with the general teachings of the invention is superior to the bispecific antibodies CD19xCD3 described so far with respect to its considerably higher biological activity as well as the possibility of its rapid and easy production, thus producing Sufficient quantities of high quality clinical grade material. Therefore, it is expected that the bscCD19xCD3 molecules of the invention are a suitable candidate for monitoring the therapeutic benefit of bispecific antibodies in the treatment of B cell-mediated diseases such as non-Hodgkin lymphoma in clinical studies. In the preferred embodiment of the polypeptide of the invention, these domains are connected to a linker polypeptide.This linker is arranged between the first domain and the second domain, wherein the linker polypeptide preferably comprises peptide-linked, hydrophilic, plural and connecting amino acids. end of the N terminal of the first domain and the end of the i.minc.i C of the second domain In a preferred embodiment of the invention the first domain and / or the second domain of the simulation of the polypeptide described above or corresponds to a VH and VL region of a natural antibody The antibody that provides the agglomerate site For the polypeptide of the invention, it can be, for example, a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a humanized protein, a bispecific antibody, a synthetic antibody, an antibody fragment such as Fv or scFv fragments that can be prepared, for example, through the techniques originally described in Kohler and Milstein, Nature 256 (1975), 495, and Galfré, Meth. Enzymol .73 (1981), 3, which comprises the fusion of mouse myeloma cells to spleen cells derived from mammals immunized with modifications developed by the technology. In addition, antibodies or fragments thereof for the aforementioned antigens can be obtained using the methods described, for example, i Harlow and Lane "Antibodies, A Manual Laboratory, "CSH Press, Cold Spring Harbor, 1988. The various organisms can be obtained from several species, including humans, when the derivatives of these antibodies are obtained through the technique of phage display, surface resonance. The same method used in the BIAcore system can be used to increase the efficiency of the phage antibodies that agglomerate an Ethiopian. -? grr-j CD19 or CD3 (Schier, Antibody Hybridomas) . '.manos 7 (1996), 97-105; Malmborg, J. Methods Immunological 183 (1995) 7-13). The production of chimeric antibodies is described, for example in the Patent WO 89/09622. Methods for the production of humanized antibodies are described, for example in - 1 '. 239 400 and WO 90/07861. An additional source of antibodies that will be used in accordance with the present invention is called xenogenic antibodies.

The general principle for the production of xenogenic antibodies such as human antibodies in mice is described, for example in WO 91/10741, WO 94/02602, , 36/34096 and WO 96/33735. The antibodies to be used according to the invention or their corresponding immunoglobulin chain (s) can also be modified using conventional techniques known in the art, for example, using the elimination (s), assertion (s). ), substitution (s), addition (s) and / or recombination (s) of amino acids and / or any other known modification in the technology either alone or in combination. The methods for introducing these modifications into the DNA sequence underlying the; The amino acid sequence of an immunoglobulin chain are well known to the person skilled in the art; see, for example, Ilambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory (1989) N.Y. : The aforementioned modification is preferably carried out at the nucleic acid level. In a further preferred embodiment of the invention, at least one of these domains in the polypeptide described above is a LL 'fragment of a single chain of a variable region of the antibody. As is well known, Fv, the smallest fragment of the antibody containing a complete recognition of the antigen and a binding site, consists of a dimer of a heavy chain and a variable light chain of the Minium (V and VL) in non-covalent association. In this configuration, which corresponds to the one found in the native antibodies, the three determining regions of e) complementation (CDR) of each of the domains or variables interact to define a binding site of the antigen on the surface of the VH dimer V__.

Collectively, the six CDRs confer their antigenic binding specificity to the antibody. The structure that The CDRs have a tertiary structure that is : 'Primarily in immunoglobulins native to species as diverse as human and mouse. These FR serve to hold the CDRs in their correct orientation. Constant domains are not required for the binder function, but they can help stabilize the VH-VT interaction. Even a simple variable i2 domain (or half of an Fv comprises only three specific CDRs for an antigen) has the ability to recognize and agglomerate the antigen, although usually with a lower affinity than that of an entire binding site (Painter, Biochem 11 '? _072), 1327-1337). Hence, this domain of the binding site of the polypeptide of the invention can be a pair of VH-VL, VH-VH or VL-VL domains either from the same immunoglobulins or from different immunoglobulins. The order of the VH and VL domains within the polypeptide chains is not decisive for the present invention, the order of the domains , previously set forth herein can usually be reversed without any loss of function. However, it is important that the VH and V domains are arranged in such a way that the binding site of the antigen can be correctly folded. In a preferred embodiment of I.JS polypeptides of this invention, the domains are arranged in the order of VLCD19-VHCD19-VHCD3 -VLC3, where y "VH" mean the light chain and the chain, > ___ > of the variable domain of anti-CD19 and anti-CD3 antibodies. As discussed above, the sites 'binders are preferably connected by a flexible linker, preferably by a polypeptide Mc-.zac.or between the domains, where the linker polypeptide comprises peptide-linked, plural, hydrophilic amino acids of sufficient length to It encompasses the distance between the end of the terminal C of one of the domains comprising these binder sites and the end of the N terminal of the other of d.unios comprising the binder sites.

(When the polypeptide of the invention assumes a L5 suitable conformation to agglomerate when it is arranged in an aqueous solution. Preferably, the linker polypeptide comprises a plurality of glycine, alanine and / or serine residues. It preferred -subditionally tthis linker polypeptide comprises Z a plurality of consecutive reproductions of an amino acid sequence. Usually, the linker polypeptide comprises from 1 to 15 amino acids although linker polypeptides of more than 15 amino acids can also work. In an embodiment 1 > Preference of the invention This linker polypeptide comprises from 1 to 5 amino acid residues. In a particularly preferred embodiment of the present invention this polypeptide linked to the polypeptide of the invention comprises 5 amino acids. As demonstrated in the examples of the index, this linker polypeptide advantageously comprises the amino acid sequence Gly Gly Gly Gly Ser. ': A particularly preferred embodiment, the first domain of the polypeptide of the invention comprises at least one CDR of the VH and VL region comprising the amino acid sequence encoded by the DNA sequence described in Figure 8 a from nucleotides 82 to 414 (VL) and nucleotides 460 to ° "1 (VH) and / or the second domain comprises at least n CDR, more preferred two, and even more preferred C r R of the V H region and VL comprising the sequence C. 1 amino acid encoded by the DNA sequence described in Figure 8 from nucleotides 847 to 1203 (VH) and nucleotides 1258 to 1575 (VL) cyonally, in combination with the regions of the child, which occurs together with CDRs in antibodies Gírenteles. The CDRs contained in the variable regions described in Figure 8 can be determined, for example, according to Kabat, "Protein Sequences of Immunological Interest" (US Department of Health and Human Services, third edition, 1983; fourth edition, 1987, fifth edition, 1990). The person skilled in the art will appreciate without difficulty tthe binder site or at least one CDR derived therefrom can be used for the construction of a polypeptide of the invention. Preferably, the polypeptide comprises the amino acid sequence encoded by the DNA sequence as described in Figure 8 from nucleotides ai 575. The person skilled in the art can readily appreciate tthe binding sites of the polypeptide of the invention can be constructed according to methods known in the art, for example, as described in EP-A1 0451 -516 and EP-A1 0 549 581. The domains of the binding sites of the polypeptide of the invention preferably have at least one specificity substantially identical to the binder specificity of, for example, the antibody or immunoglobulin chain from which they were derived. These domains of the binding site may have agglomeration affinity of at least 105M ^ 1, preferably not greater than 107M_1 for the CD3 antigen and advantageously up to 1010M "1, or higher for the CD19 antigen In a preferred embodiment of the polypeptide of the invention: (a) the binder site of the first domain has an affinity of at least of the order of 10"7M, preferably of at least of the order of 10 ~ 9M and more inverse of at least of the order of 10_11M; I (b) the binding site of the second domain has an affinity of less than the order of 10"7M, - preferably of less than the order of 10 ~ 6M and more preferably of the order of 10 ~ 5M. According to the preferred embodiments referred to above, it is advantageous if the binding site which recognizes the CD19 antigen has a high affinity in order to capture the cells > Lanco that must be destroyed with high efficiency. On the other hand, the agglomerating affinity of the binding site which recognizes the CD3 antigen must be the order of those of the natural CD3 receptor or that which is usually found for the interaction of the T cell receptor with its ligament, which is a complex of MHC peptide on the surface of the target cell. In another preferred embodiment of the invention, the polypeptide described above is my bispecific single chain antibody.

The present invention additionally relates to a polypeptide comprising at least one additional minie, these domains are linked by covalent or non-covalent bonds. The link can be based on a genetic fusion according to the methods known in the art and described above or that can be performed, for example, through a chemical translation as described, for example in, i? WO 94/04686. The additional domain present in the polypeptide of the invention can preferably be linked by a flexible linker, advantageously a polypeptide linker to one of the domains of the binding site where the linker polypeptide comprises Molecules attached to the plural, hydrophilic peptide of ana longitude to encompass the distance between the terminal C terminus of one of the domains and the N terminal end of the other domain when the polypeptide assumes a suitable conformation to agglomerate when available in a solution Preferably, this linker polypeptide is LUÍ polypeptide linker as described above in the embodiments of the present. The polypeptide of the invention may further comprise a fracture linker or a fracture site for proteinases, such as enterokinase; see also the examples in the appendix. In addition, the additional domain may have a predefined function or • specificity. For example, the Iterature contains a host of references on the concept of targeting bioactive substances such as drugs, toxins, and enzymes to specific points in the body to destroy or localize malignant cells or to induce a localized drug effect. or enzymatic. It has been proposed to achieve this effect by conjugating the bioactive substance to the monoclonal antibodies (see, for example, Oxford University Press and Ghose, J. Nati, Cancer Tnst 61 (1978), 657-676). In the context, it is also understood that the polypeptides according to the invention can also be modified by conventional methods known in the art. This allows the construction of chimeric proteins comprising the polypeptide of the invention and other functional sequences of the amino acid, for example, nuclear localization signals, transactivating domains, DNA binding domains, hormone binding domains, labeled protein (GST, GFP, peptide h-myc, 1-1LAG, peptide HA) that can be derived from heterologous proteins. As described in the examples of the appendix, the polypeptide of the invention comprises "> -efferently a FLAG tag of about 8 amino acids long, see Figure 8. The polypeptides of the invention can be therapeutically ucilized in patients suffering of B-cell disorders such as lymphoma of the cell or chronic lymphatic leukemia derived from the B \.-CLL cell) and / or having an autoimmune disease Ll related to B cell as myasthenia gravis, Morbus Basedow, Hashimoto thyroiditis, or 'iod.pasr.ure syndrome. This therapy can be achieved through, for example, the administration of the polypeptides of the invention. This administration can use unlabeled polypeptides as well as labeled polypeptides. For example, the polypeptides of the invention can be administered labeled with a therapeutic agent.These agents can be coupled directly or indirectly to the antibodies or antigens of the invention.An example of an indirect coupling is through the use of a spacer half.

These spacer moieties, in turn, may be soluble or soluble (Diener, Science 231 (1986), 148) _ may be selected to enable drug release of the antigen at the target site. The effective therapeutic examples that can be coupled to the polypeptides of the invention for immunotherapy are drugs, radioisotopes, lectins and toxins. Drugs conjugated to the polypeptides of the invention include compounds that are classically referred to as drugs such as mitomycin C, .norubicin and vinblastine. In the use of radioisotopically conjugated polypeptides of the invention, for example, immunotherapy, hundreds of isotopes may have greater preference than others depending on factors such as the distribution of leukocyte as well as stability • mirr.ón Depending on the autoimmune response, some issuers may be preferred over others. In general, particle emitting radioisotopes a and ß are preferred in immunotherapy. Those of preference are the emitters to short attack, high energy like 212Bi. Examples of radioisotopes can be linked to the polypeptides of the invention for therapeutic purposes are 125 I, 131 I, 90 Y, 67 Cu, 212 Bi, 212 A t, 211 Pb, 47 S c, 109 P d and 188 Re. Lectins are proteins usually isolated from plant material, which agglomerate to specific sugar moieties. Many lectins also have the ability to agglutinate cells and stimulate lmfocytes. However, ricin is a toxic lectin that has been used immunotherapeutically. This is achieved by agglomerating the chain of peptide a from 5 ± cin, which is responsible for the toxicity, to the lipeprid to allow the specific delivery in the itio of the toxic effect. Toxins are poisonous substances produced t plants, animals, or microorganisms that, in doses 'lucid, they are often lethal. The toxin i phtpa is a substance that is produced by the , 'and-rinebacterium diphtheria that can be used therapeutically. This toxin consists of a subunit a and ß that under the correct conditions i r can be separated. The toxic component A can be linked to a polypeptide of the invention and used for site-specific delivery to interact r > n the B cell and the T cell that have been placed in close proximity through an agglomeration towards a polypeptide of the invention. Other therapeutic agents such as those previously written that can be coupled to the polypeptide of the invention, as well as correspond to therapeutic protocols ex vi ve and in vi ve, are known, or can be easily established, by those persons who are ordinarily trained. in the technology. Whenever correct, the person skilled in the art can use a polynucleotide of the invention described below > present, coding for any of the polypeptides described above or the corresponding vectors in place of the proteinaceous material. Thus, the person skilled in the art can appreciate without problems that the polypeptide of the invention can be used for the construction of other polypeptides of specificity .. seada and biological function. It is expected that the polypeptides of the invention play a therapeutic Important and a scientific role in particular in the medical field, for example, in the development of new treatment approaches for disorders associated with B cells such as certain forms of cancer and autoimmune diseases or as interesting tools for the analysis and modulation of transduction pathways of the corresponding cellular signal. In a further preferred embodiment of the invention, at least one of the Additional minios comprises a molecule selected from the group consisting of effector molecules having a conformation suitable for biological activity, amino acid sequences capable of sequestering an ion, and amino acid sequences capable of selectively agglomerating to a solid support or a preselected antigen . Preferably, additionally this domain comprises an enzyme, a toxin, a receptor, a binding site, a binding site of biosynthetic antibody, a remotely detectable moiety, an antimetabolite, a radioactive atom or an antigen. i.O This antigen can be, for example, a tumor antigen, a viral antigen, a microbial antigen, an allergen, an autoantigen, a virus, a microorganism, a polypeptide, a peptide or a plurality of tumor cells. In addition, this sequence capable of sequestering an ion is preferably selected from a calmodulin, a metallothiolein, a functional fragment thereof, or an amino acid sequence rich in at least one glutamic acid, an asparatic acid, lysine or arginine. . Additionally, the polypeptide sequence has the ability to selectively agglomerate to a solid support which may be a positively or negatively charged amino acid sequence, a I b cysteine containing an amino acid sequence, avidin, streptavidin, a functional fragment of the -Try Staphylococcus A, GST, a His tag, a quet i FLAG or Lex A. As described in -appendix examples, the polypeptide of the invention that is exemplified by a single chain antibody has also been expressed with an N-terminal FLAG tag and / or a C-tag His tag which allows for easy purification and detection. The tag FLAG yielded in the example comprises 8 amino acids (see lag 8) and is thus preferably used according to the present invention. However, FLAG tags comprise shortened versions of FLAGs used in the examples of the appendix as the amino acid Asp-Tyr-Lys-Asp cue that are also available. The effector molecules and the amino acid sequences described above can be present in a proforma that is itself active or not and that can be removed, when, for example, they enter a certain cellular environment. In a preferred embodiment of The invention, the receptor is a molecule of a stimulatory surface important for the activation of the T cell or comprises an epitope binding site or a hormone binding site.

In a further preferred embodiment of the present invention, the molecule of the costimulatory surface is CD80 (B7-1) or CD86 (B7-2). Still, in a further embodiment, the present invention relates to polypeptides which, in the presence of expression, encode the polypeptides described above. These polynucleotides can be fused with control sequences of a suitable expression known in the oenology to ensure correct transcription and transfer of the polypeptide. This polynucleotide may be, for example, DNA, cDNA, RNA or synthetically produced DNA or RNA or a recombinantly produced chimeric nucleic acid molecule comprising any of The polynucleotides either alone or in combination.

Preferably, the polynucleotide is part of a vector. These vectors may further comprise genes such as gene makers that allow selection of the vector in a suitable host cell and under suitable conditions. Preferably the "Thiolinucleotide of the invention is operatively linked to the expression control sequences allowing expression in prokaryotic or eukaryotic cells." Expression of the polynucleotide (undertakes transcription of the polynucleotide in a translatable "RN to mRNA." The regulatory elements ensure the expression in eukaryotic cells, preferably mammary cells, are well known to those skilled in the art, these usually comprise regulatory sequences assuring the initiation of transcription and ip poly-signaling ensuring the termination of transcription and the stabilization of transcription. Additional regulatory elements may include transcriber enhancers as well as translators and / or naturally associated regions or heterologous promoters Possible regulatory elements allow expression in prokaryotic host cells comprising, for example, or, the PL, lac, trp or tac promoter in E. coli, and examples of regulatory elements that allow expression in eukaryotic host cells are the A0X1 or AGAL1 promoter in yeast or the CMV promoter, SV40, RSV ( Rous sarcoma), CMV enhancer, SV40 enhancer or a globin intron in mammary cells and in other animal cells. Lateral elements that are also responsible for the initiation of transcription as regulatory elements may also comprise the inscription-termination signals, such as the SV40-poly-A, downstream "1-poiinucleotide site. Furthermore, depending on the expression system used the leader sequences have the ability to direct the polypeptide to a cellular compartment or secrete therein the medium that can be added to the coding sequence of the polynucleotide of the invention and which is well known in the art; see also, for example, the examples in the appendix. The leader sequence (s) is coupled in the correct phase with the translation, initiation and termination sequences and preferably, a leader sequence capable of directing the secretion of the -translated protein, or a portion thereof, in a periplasmic or extracellular space. Optionally, the heterologous sequence can encode a fusion protein, including an N-terminal identification peptide imparting the desired characteristics, eg, tabilization or simplified purification of the expressed recombinant product; see above In this context, suitable expression vectors are also known in the technology as Okayama-Berg expression vector cDNA pcDCl (Pharmacy), pCDM8, pRc / CMV, pcDNA, pcDNA3 (in-vi trogene), or pSPORT 1 (GIBCO BRL ). Preferably, the expression control sequences will be eukaryotic promoter systems in the vectors capable of transforming the eukaryotic host cells that are transferred, but the control sequence for the prokaryotic hosts can also be used. Once the vector has been incorporated into the correct host, the host is maintained under conditions suitable for high level expression of the nucleotide sequences, and as desired, the collection and purification of the polypeptide of the invention can follow; see, for example, the examples in the appendix. As described above, the polynucleotide of the invention can be used alone or as part of a vector to express the polypeptide of L_ the invention in cells, for example, for gene therapy or diagnoses of diseases related to disorders of the B cell. The polynucleotides or vectors containing the DNA sequence (s) encoding any of the ? The polypeptides described above are introduced into the cells which in turn produce the polypeptide of interest. Gene therapy, which is based on the introduction of therapeutic genes into the cells through techniques ex vi vo or in vi vo is one of the 5 most important applications of gene transfer. The appropriate vectors, gene delivery methods or systems for gene therapy in vi tro or in vi vo are described in the literature and are known by the • arsona-enabled technology, see, for example, erdape, Nature Medicine 2 (1996), 534-539; Scharper, LLGC. ...is. 79 (1996), 91 | 1-919; Anderson Science 256 (1992), 808-813; Verma, Nature 389 (1994), 239; Isner, Lancet 348 (1996), 370-374; Muhlhauser, Circ. Res. Yy (1995), 1077-1086; Onodera, Blood 91 (1998), 30.36; 'erma, Gene Ter. 5 (1998), 692-699 Nabel, Ann N.Y. • ad. , ci. 811 (1997), 289-292; Verzeletti, Hum. Gene ner. 9 (1998), 2243-51; Wang, Nature Medicine 2 (1996), 714-716; WO 94/29469; WO 97/00957, US ,580,859; US 5,589,466; o Schaper, Current Opinion in Biotechnology 7 (1996), 635-640, and cited references In the same. The polynucleotides and vectors of the invention can be designated for direct introduction or for introduction through liposomes, or viral vectors (eg, adenoviral, retroviral) or into the cell. Preferably, this cell is a germ line cell, an embryonic cell, an egg cell or derivatives thereof, more - said cell is preferably a stem cell. An example of an embryonic stem cell may be, among other things, a stem cell as described in, Nagy, Proc. Nati Acad. Sci. USA 90 (1993), 8424-8428. According to the above, the present invention relates to vectors, particularly plasmids, cosmids, viruses and bacteriophages conventionally used in genetic engineering comprising the polynucleotide encoding a polypeptide of the invention. Preferably, this vector is an expression vector and / or a transfer of target typing gene or vector. Expression vectors derived from viruses such as retroviruses, vaccinia virus, adeno-associated virus, herpes virus, or bovine papilloma virus can be used to deliver the polynucleotides or the vector of the invention to target cell populations. The methods that are well known to people skilled in the technology can be used to construct recombinant vectors; see, for example, the techniques described in Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory (1989) N.Y. and Ausubel, Current Protocols. Molecular Biology, Green Publishing Associates, and Wiley Interscience, N.Y. (1989). Alternatively, the polynucleotides and vectors of the invention can be reconstituted in liposomes to be delivered to target cells. The vectors containing the polynucleotides of the invention can be transferred into the host cell by well-known methods, which vary depending on the type of cellular host. For example, calcium chloride transfection is commonly used for prokaryotic cells, where calcium phosphate treatment or electroporation can be used for other cellular hosts; see Sambrok, supra. Once expressed, the polypeptide of the present invention can be purified according to the standard procedures of the technology, including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis and the like; see, 1 Lcances, "Purification of Protein", Springer-Verlag, N.Y. / 1982). Substantially pure polypeptides of at least about 90 to 95% homogeneity are preferred, and even more preferred in the order of 98 to 99% or more of homogeneity, for pharmaceutical uses. Once purified, partially or at the desired homogeneity, the polypeptides can then be used therapeutically (including extracorporeally) or in the development and performance of assay procedures. In yet another embodiment, the present invention relates to a cell that contains the polynucleotide or vector described above. Preferably, this cell is a eukaryotic, more preferably a mammary cell, or one that provides for therapeutic uses of the polypeptide. Of course, less preferred yeast and prokaryotic, for example bacterial cells may also serve, in particular if the polypeptide produced is used as a diagnostic element. The polynucleotide or vector of the invention that is present in the host cell can be integrated into a genome of the host cell or can be extra chromosomally maintained. The term "prokaryotic" means that it includes all bacteria that can be transformed or transfected with DNA or RNA molecules for the expression of a polypeptide of the invention. Prokaryotic hosts may include gram negative or gram positive bacteria such as, for example, E. coli, S. tifimurium, Serratia marcescens and Bacillus subtilis. The term "eukaryotic" means that it includes Yeast, upper plant, insect cells and preferably mammary cells. Depending on the host employed in the recombinant production process, the polypeptides of the present invention can be glycosylated or non-glycosylated. The polypeptides of the invention may also include an initial methionine amino acid residue. A polynucleotide encoded for a polypeptide of the invention can be used to transform and transfect the host using any of the techniques commonly known to those skilled in the art. The use of a plasmid or a virus containing the coding sequence of the polypeptide of the invention and genetically fusing it is especially preferred. n a terminal N label FLAG and / or terminal C label His. Preferably, the length of the FLAG tag is of the order of 4 to 8 amino acids, more preferably of 8 amino acids. The methods for preparing fused genes operably linked and expressed in, for example, mammary cells and the '> and Ctenas are well known in technology (Sambrook, Molecular Information: A Laboratory Manual, Cold, The Ring Harbor Laboratory, Cold Spring Harbor, N.Y. 1989). The constructs and genetic methods described therein can be used for the expression of the polypeptide of the invention in eukaryotic or ochapotic hosts. in general, the expression vectors < The promoter sequences that facilitate efficient transcription of the inserted polynucleotide are made in connection with the host. The expression "vector" typically contains a replication origin, a promoter and a terminator, as well as specific genes that have the ability to provide a phenotypic selection of the transformed cells. In addition, transgenic animals, preferably mammals, comprise cells of the invention can be used for large-scale production of the polypeptide of the invention. invention. In a further embodiment, the present invention further relates to a method for the preparation of a polypeptide described above which comprises culturing a cell of the invention under conditions suitable for the expression of the polypeptide and isolating the polypeptide from the cell or from the culture medium. Transformed hosts can grow in termenters and be cultivated according to techniques * s, known in technology to achieve optimal cell growth. The polypeptide of the invention can then be isolated from the growth medium, cellular, or cell membrane fractions. The isolation and purification of, for example, The microbially expressed oligopeptides of the invention can be through any conventional element such as, for example, chromatographic preparative separations and immunogenic separations such as those involving the use of monoclonal or polyclonal antibodies directed, for example, against a polypeptide tag of the invention or as < - e described in the appendix examples. Thus, the present invention allows the recombinant production of the polypeptides that comprise binder sites having affinity and specificity for epitope of the CD19 and CD3 antigen, respectively and optionally an additional functional domain. As is evident from the above, the invention provides a long family of polypeptides comprising these binder sites for any use in therapeutic and diagnostic approaches. It will be apparent to those skilled in the art that the polypeptides of the invention can additionally be coupled with other moieties as described above for example, for drug target fixation and imaging applications. These couplings can be chemically driven after the expression of the polypeptides at the site of adhesion or coupling of the product can be engineered into the polypeptide of the invention at the DNA level. The DNAs are then expressed in a suitable host system, and the expressed proteins are harvested and renatured, if necessary. As described previously, the binder sites are derived -efferently of the variable region of the antibodies. In this respect the hybridoma technology allows the production of the antibody that secretes cell lines to essentially any desired substance that produces an immune response. He TrN that encodes the light and heavy chains of the '.muno? 1 obulin can be obtained from the cytoplasm of the hybridoma. The final 5 'portion of the mRNA can be used to make the cDNA for use in this method of the present invention. He DNA encoding the polypeptides of the invention can be expressed expressly in cells, preferably mammary cells. Depending on the host cell, renaturation techniques may be required to obtain the correct conformation. If necessary, point substitutions that seek to optimize the , Lomeration can be performed on the DNA using conventional cassette istagenesis or other engineering methodology of the protein as revealed in the present. The preparation of the polypeptides of the invention may also depend on the knowledge of the amino acid sequence (or the corresponding DNA or RNA sequence) of the bioactive proteins such as enzymes, toxins, growth factors, cell differentiation factors. , antibodies, antimetabilites, hormones or various cytokines or lymphokines. These sequences are reported in the literature and are available through computerized data banks. For example, a polypeptide of the invention can be constructed, which, for example, consists of a single chain Fv fragment and the extracellular part of the human CD50 (B7-1) imulatory cost protein connected by a linker (Gly4Serl) I. The CD80 imulatory cost protein belongs to the super Ig family. It is a heavily glycosylated protein of the 262 amino acids. A more detailed description was published by Freeman, J. rmmunol. 143 (1989), 2714-2722. Stable expression can be performed, for example in CHFR deficient CHO cells described by Kaufmann, Methods Enzymol. 185 (1990) 537-566. The protein can then be purified through its His tag attached to the C-terminus using a Ni-NTA column (Mack)., Proc. Nati Acad. Sci. U.S.A. 92 (1995), 7021-7025). Additionally, the present invention provides L LS compositions comprising the aforementioned polypeptide, the polynucleotide or the vector of the invention. Preferably, the present invention relates to compositions that are pharmaceutical compositions comprising the aforementioned polypeptide (s), polynucleotide (s) or vector (s) of the invention. The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier. Examples of suitable pharmaceutical carriers are well known in the art and include buffered saline solutions of phosphate, water, emulsions, such as oil / water emulsions, various types of wetting agents, sterile solutions, etc. The compositions comprise the vehicles that can be formulated by conventional well-known methods. These pharmaceutical compositions can be administered to the subject in a suitable dose. The appropriate administration of the compositions can be carried out in different ways, for example, intravenously, intraperitoneally, subcutaneously, intramuscularly, topically and by intradermal administration. The dose regimen will be determined by attending the doctor and clinical factors. As is well known in medical technologies, the doses to any patient depend on many factors, including the patient's size, the area of the body surface, age, the particular compound to be administered, sex, time and route. of administration, general health, and other drugs are administered concomitantly. Generally, the ... regimen as a regular administration of the pharmaceutical composition should be in the range of 1 μg to 10 mg units per day. If the regimen is a continuous infusion, it should also be in the range of 1 μg to 10 mg of units per kilogram of body weight per minute, respectively. However, a dose , s preferred for continuous infusion can be in the range of 0.01 μg to 10 mg units per kilogram of body weight per hour. Particularly preferred doses are described below. Progress can be monitored through periodic assessment. Doses will vary but a dose by intravenous saininictration of DNA is from approximately 106 to 1012 replications of the DNA molecule. The compositions of the invention Pi to be administered locally or systematically. The administration will generally be parenteral, for example, intravenously; the DNA can also be administered directly to the target site, for example 5 through biologic delivery to an internal or external target site or via catheter to a site in an artery. Preparations for parenteral administration include sterile aqueous solutions or non-aqueous solutions, suspensions, and emulsions. The Examples of the non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters and ethyl oleate. Aqueous vehicles include water, alcoholic / aqueous solutions, L5 emulsions or suspensions, including saline and buffered media. Parenteral vehicles include a solution of sodium chloride, Ringer's dextrose, dextrose and sodium chloride, Ringer's lactate, or combination oils. Intravenous vehicles 0 include fluid and nutrient fillings, electrolyte fillings (such as those based on Ringer's dextrose), and the like. Also present are preservatives and other additives such as, for example, antimicrobials, antioxidants, agents to laminators, inert gases and the like.

Additionally, the pharmaceutical composition of the This invention may include oteinaceous vehicles, such as, for example, serum albumin, or immunoglobulin, preferably of human origin.

Furthermore, it is envisaged that the pharmaceutical composition of the invention may further comprise biologically active agents, depending on the intended use of the pharmaceutical imposition. These agents can be Drugs that act on the gastrointestinal system, drugs that act as cytostatic drugs, drugs to prevent hyperuricemia and / or agents such as T cell costimulatory molecules or cytokines known in the technology. It is envisioned by the present invention that < The polynucleotides and vectors of the invention are administered alone or in any combination using standard vectors and / or gene delivery systems, and optionally together with acceptable pharmaceutical carriers or excipients. Subsequent to the modification, the polynucleotides or vectors can be stably integrated into the genome of the subject. On the other hand, viral vectors that are specific for certain cells or tissues and persist in these cells can be used. Suitable pharmaceutical carriers and excipients are well known in the art. The pharmaceutical compositions prepared according to the invention can be used for the prevention or treatment or delay of different types of diseases, which are related to the immunodeficiencies and malignities related to the B cells. Furthermore, it is possible to use a pharmaceutical composition of the invention comprising the polynucleotide or the vector of the invention in gene therapy. Suitable gene delivery systems may include liposomes, delivery systems measured by the recipient, naked DNA, and viral vectors such as herpes viruses, retroviruses, adenoviruses, and adeno-associated viruses, among others. Delivery of nucleic acids to a specific site in the body for gene therapy can also be carried out using a biolistic delivery system, such as that described by Williams (Proc. Acad. Sci. USA "8 (1991), 2726 -2729). Additional methods for nucleic acid delivery comprise particle-mediated gene transfer, as described, by Eg in Verma, Gene Ther.15 (1998), 692-699. It should be understood that the polynucleotides and the introduced vectors express the gene product after introduction into the cell and preferably remain in this state for the life of the cell. For example, the cell lines stably expressing the polynucleotide under the control of the correct regulatory sequences can be engineered according to the well-known methods of the IcyS-enabled persons in the technology. In using the expression vectors containing viral replication origins, the host cells can be transformed with the polynucleotide of the invention and with a selectable marker, either in the same plasmids or in plasmids separately. If the introduction of foreign DNA is allowed, engineered cells can be allowed to grow for 1-2 days in an enriched medium, and then switch to a selective medium.

The selectable marker in the recombinant plasmid confers resistance to selection and allows the choice of cells that have stably integrated the plasmid in their chromosomes and that grow to form foci that in turn can be cloned and expanded within the cell lines. These engineered cell lines are also • particularly useful in filtering methods for the detection of the compounds involved in, for example, the interaction of the B-cell / T-cell.

You can use a number of systems -election, including, but not limited to, the herpes simplex virus thymidine kinase (Wigler, Cell 11 (1977), 223), guanine phosphoribosyltransferase hypoxanthine (Szybalska, Proc. Nati. Acad. Sci. USA 48 (1962), 2026), and adenine phosphoribosyltransferase , owy, Cell 22 (1980), 817) in the tk ", hgprt" p aprt "cells, respectively. Also, the resistance of the antimetabolite can be used as the gbase for the The selection of dhfr, which confers resistance to methotrexate (Wigler, Proc, Nati, Acad. Sci. USA 77 (1980), 3567; O'Hare, Proc. Nati Acad. Sci. USA 78 C 981), 1527), gpt, which confers resistance to mycophenolic acid (Mulligan, Proc. Nati, Acad. Sci. USA 78 5 (1981), 2072); neo, which confers resistance to ammoglycosides F-418 (Colberre-Garapin, J. Mol. Biol. 150 (1981), 1); hygro, which confers resistance to hygromyme (Santerre, Gene 30 (1984), 147); or iromycin (pat, puromycin N-acetyl transferase). Additional selectable genes, for example troB, have been described, which allow cells to use a type instead of tryptophan, hisD, which allows cells to use histinol instead of histidine.

(Hartman, Proc. Nati, Acad. Sci USA 85 (1988), 8047); Y '"ODC (ornithine decarboxylase) which confers resistance to the ornithine decarboxylate inhibitor, 2- (difluoromethyl) -DL-ornithine, DFMO (McCologue, 1987, current Communications in Molecular Biology, Cold Spring Harbor Laboratoru, ed.). In another embodiment, the present invention relates to a diagnostic composition comprising any of the polypeptides, polynucleotides or vectors of the invention described above and which are optionally suitable means for detection. The polypeptides of the invention are also suitable for use in immunoassays where they can be used in the liquid phase or combined with a carrier in the solid phase. Examples of these immunoassays that can use the polypeptide of the invention are competitive and non-competitive immunoassays both in direct format and in indirect format. Examples of these immunoassays are the radioimmunoassay (RIA), the "sandwich" (immunometric assay) and the western blotting assay "Western blot". The polypeptides of the invention can be combined with different carriers and used to isolate specifically combined cells to these polypeptides. Examples of well-known vehicles include, glass, polystyrene, polyvinyl chloride, polypropylene, polyethylene, polycarbonate, dextran, nylon, amyloses, natural and modified celluloses, colloidal metals, polyacrylamides, • arosp, and magnetite. The nature of the vehicle may be soluble or insoluble for the purposes of the invention. There are many labels and many different labeling methods known to people ordinarily skilled in technology. Lugs of these types of labels that may be used in the present invention include enzymes, radioisotopes, colloidal metals, fluorescent compounds, chemiluminescent compounds, and bioluminescent compounds; see also the embodiments discussed above in the present. The present invention also relates to the use of the polypeptide, the polynucleotide, and the vector of the invention described hereinabove for the preparation of a pharmaceutical composition for the treatment of B cell malignancies, autoimmune diseases mediated by the B cell. or the depletion of B cells. Recent clinical studies with cytotoxic activity again targeting human Pte T cells through bispecific antibodies have shown promising results in the treatment of refractory Hodgkin disease (33) breast and ovarian cancer (34-37) and malignant glycoma (38). Given the facts that bsc antibodies due to their low molecular mass facilitate penetration into tumors (as demonstrated by Fab or Fv fragments) (36); and - that bsc antibodies are suspected to decrease the dose-dependent and dose-limiting toxicity caused by the cytosine release mediated by the Fc portions of conventional bispecific antibodies (40); and - that still an intact monoclonal antibody (directed against CD20) led to regression of the tumor in the advanced stages of NHL (41, 42), it is expected, and in fact has been demonstrated, that the polypeptides of the invention are interesting molecules that contribute to further therapeutic improvements. Thus, in a preferred embodiment, the pharmaceutical composition of the invention is used for the treatment of non-3S lymphoma of the Hodgkin type.

The ranges of the administration dose of the polypeptides, the polynucleotides and the vectors of i. invention are those large enough to produce the desired effect in which the symptoms of the disease mediated by B cells improve satisfactorily. The dose should not be so large that it could cause essential adverse side effects, such as unwanted cross-reactions, anaphylactic actions, and the like. Generally, the dose will vary with the age, condition, sex and extent of the disease in the patient and can be determined by a person skilled in the technology. The dose can be adjusted by the individual's doctor in the case of any contraindication. The range of the dose is adjusted to, for example, 0.01 μg to 10 mg of the polypeptide of the invention. A particularly preferred dose is 0.1 μg to 1 mg, even more preferred is 1 μg to 100 μg and more preferably is a dose of 3 μg to 10 μg as, for example, illustrated in example 7 of the .. . In addition, the invention relates to a method for identifying the C cell that activates or co-stimulates the compounds or to identify the T cell activation and stimulation inhibitors comprising (a) the culture of CD19 positive cells (preferably B cells) and T cells in the presence (the polypeptide of the invention and, optionally, in the presence of a component capable of providing a detectable signal in response to activation of the T cell with a compound that is filtered under conditions that allow interaction of the compound with the cells; and (b) detecting the presence or absence of a pitted signal from the interaction of the compound with the cells.This embodiment is particularly useful for testing the ability of the components as 5 costimulatory molecules.In this method, the CD positive cell 19 / cell B provides a primary activation signal for the T cell, this is how the conotypic T-cell receptor is prevented. Then, it can be determined according to the invention which compound 0 to be tested is still necessary to actually activate the T cell. In the method of the invention, The positive cell CD19 / cell B functions as a stimulating cell that links the bispecific molecules that bind to the CD3 complexes in : '• The surface of the same T cell. The biological methods to carry out the culture, the detection and optionally, the proof are clear to a person enabled in the technology. The term "compound" in the method of the invention includes a single substance or a plurality of 2 substances which may or may not be identical. The compound (s) may (eg) comprise, for example, samples, for example, from cell extracts of, for example, plants, animals or microorganisms. In addition, these compounds may be known in the art but until now they are not known to have the ability to inhibit T cell activation or are not known to be useful as a costimulant factor of the T cell, respectively. The plurality of the compounds can be, for example, added to the culture medium or injected into another cell. If a sample containing a tax (s) is identified in the method of the invention, then it is possible to isolate the compound from the original sample identified as containing the compound in question, or the original sample can be further subdivided, for example, if it consists of a plurality of different compounds, in such a way that . Determine the number of different substances per sample and repeat the method with the subdivisions of the original oyster. It can be determined whether said composite sample exhibits the desired properties through the methods known in the art as described herein and in the examples of the appendix. Depending on the complexity of the samples, the steps described above may be performed three times, preferably until the sample identified according to the method of the invention only comprises a limited number or only one substance (s). Preferably the sample comprises substances or similar chemistry and / or physical properties, and more preferably these substances are identical. The methods of the present invention can be easily performed and designed by the person skilled in the art, for example, according to another cell based assay described in the prior art or through using and modifying the methods as described in the examples of the index. Furthermore, the person skilled in the art will recognize without problems which are the compounds and / or additional cells that he will use in order to carry out the methods of the invention, for example, interleukins, or enzymes, if necessary, that convert a certain compound in the precursor that in turn stimulates or suppresses the activation of the T cell.

This adaptation of the method of the invention is within the capacity of the person empowered in the oenology and can be carried out without undue experimentation. The compounds that can be used according to the method of the present invention include peptides, proteins, nucleic acids, antibodies, small organic compounds, ligatures, peptide replication, APN and the like. These compounds may also be functional derivatives or analogs of the T cell activators or inhibitors. Methods for the preparation of chemical derivatives and the like are well known to those skilled in the art and are described, for example, in Beilstein, • aderno de Química Organica, Springer edition New York, Inc. 175 Fifth Avenue, New York, NY 10010 E.U.A. and Organic Synthesis, Wiley, New York, USA. In addition, derivatives and analogs can be tested for their effects according to the methods known in the art as described, for example in the examples of the appendix. In addition, the peptide reproductions and / or the computer-aided design of the correct activators or inhibitors of T cell activation can be used, for example, according to the methods described below. Correct computer programs can be used for the identification of interactive sites of a putative inhibitor and the antigen of the invention through computer-assisted searches for complementary structural motifs (Fassina, Immunomethods 5 (1994), 114-120). In addition, the correct computer systems for the computer-aided design of protein and peptides are described in the prior art, for example, in Berry, Biochem. Soc. Trans. 22 (1994), 1033-1036; Wodak, Ann. N.Y. Acad. Sci. 501 (1987), 1-13; Pabo, Biochemestry 25 (1986), 5987-5991. The results obtained from the computer analysis described above can be used in combination with the method of the invention for example, to optimize the known activators or inhibitors of T-cell. The replicas of correct peptides can also be identified through the synthesis of combined libraries of peptide replicates through successive chemical modification and testing the resulting compounds, for example according to the method described herein and in the examples of the appendix. Methods for the generation and use of the combined libraries of the peptide replicas are described in the prior art, for example, in Ostresh, Methods in Enzymology 267 (1996), 220-234 and Dorner, Bioorg. Med. Chem. 4 (1996), 709-715. In addition, the three-dimensional and / or crystallographic structure of the inhibitors or activators of the B cell / T cell interaction can be used for the design of inhibitors or activators of the peptide replication of the T cell activation that will be tested in the method of the invention (Rose, Biochemistry 35 (1996), L2933-i2944; Rutenber, Bioorg. Med. Chem. 4 (1996), 1545-1558). In summary, the present invention provides methods for identifying compounds that have the ability to modulate the measured immune responses of the B cell / T cell. The compounds found to activate the B cell / T cell mediated responses can be used in the treatment of cancer and related diseases. Additionally, it may also be possible to specifically inhibit viral diseases, thus preventing viral infection or viral propagation. Compounds identified as suppressors of activation or stimulation of the T cell can be used in the -an transplant of organs in order to avoid rejection of the graft; see also above.

In this way, it is expected that the compounds or compounds obtained according to the method of the present invention will be very useful in diagnosis and in particular for therapeutic applications. Hence, in a further embodiment the invention relates to a method for the production of a pharmaceutical composition comprising the formulation of the compound identified in step (b) of the methods described above of the invention LO in a pharmaceutically acceptable form. In addition, it is anticipated that this component can be modified by peptide replicates. The methods for the generation and use of combined libraries of optical replicas are described in the prior art, for example in Ostrech, Methods of Enzymology 267 (1996), 210-234, Doner, Bioorg. Med. Chem. 4 (1996), 709-715, Beeley, Trends Biotechnol. 12 (1994), 213-216, or al-Obeidi, Mol. Biotech 9 (1998) 205-223. Therapeutically useful compounds disclosed in accordance with the method of the present invention can be administered to a patient through any correct method for the particular compound, for example, orally, intravenously, parenterally, transdermally, transmucosally, or go surgery or implant (for example, with the compound in the form of a solid or semi-solid matrix, biologically compatible and resorbable) in or near the guy where the effect of the compound is desired. The therapeutic doses are determined as correct by a person qualified in technology, see above. Additionally, the present invention provides a method for the treatment of B cell malignancies, autoimmune diseases mediated by B cell or B cell depletion and / or a method that delays a pathological condition that is caused by disorders of the B cell comprising the introduction of the polypeptide, the polynucleotide or the vector of the invention into a mammal affected by these malignancies, this disease and / or pathological condition. it is also preferred that the mammal be a human. These and other embodiments are disclosed and accompanied by the description and Examples of the present invention. Additional literature concerning any of the antibodies, methods, uses and compounds to be employed in accordance therewith; ivención can be recovered from public libraries and data oases, using for example electronic devices. For example, the public database "Medline" which is available on the Internet can be used, for example under h - t: // www .ncbi .nlm.nih. gov. / PubMed / medline. html The J-data and address, such as http: // www. ncbi. nlm. nih gov /, http: // www. infobiogen. fr /, tp: /, ww. fmi. ch / bilogy / research_tools. html, Lttp: // www. tigr org / are known to the person in technology and can also be obtained using, for example, http://www.lycos.com. A perspective of patent information in biotechnology and a survey of relevant resources and patent information useful for retrospective research and for current awareness is given in P.erks, TIBTECH 12 (1994), 352-364.

The figures show: Figure i. : SDS page: Coomassie blot of purified bscCD19xCD3 fragment with different amounts of protein. The molecular mass (kDa) of the marker is; idica on the left. .cura:: The FACS analysis with the bscCD19xCD3 (200 μg / ml) in different lines of the positive B cell CD19 (BJAB,? KW6.4, Blin-1, Daudi, Raji), in the negative B cell line CD19 BL60 and in primary Jurkat CD3-positive human PBMC cells. Dotted lines indicate negative controls. Figure: Cytotoxicity of bscCD19xCD3 in a 51Cr release assay with unstimulated human PBMC and different B cell lines. Relation of the cell between 1 rector - White 10: 1; incubation time 4 hours. standardized in all triplicates was below 7%. 'igura' í: The chromium release cytotoxicity assay with Primary human PBL without stimulating against the lines of Plasmacytoma cell L363 and NCI and line of the Daudi lymphoma cell with E: T ratio of 20: 1; incubation time 8 hours. 'cure: Inhibition of the cytotoxicity of bscCD19xCD3 by the anti-CD19 HD37 parental antibody in a chromium release assay; incubation time 8 hours; E: T ratio of 20: 1; bscCD19xCD3 concentration of lng / ml. Figure 6: Cytotoxicity assay with PBMC without stimulating against Daudi cells after addition of increasing amounts of EGTA, E: T ratio of : 1, incubation time 4 hours. Figure 7: whether cytotoxicity of bscCD19xCD3 in a 51Cr release assay with unstimulated human PBMC and Blin-1 as target cells at different E: T ratios; incubation time 4 hours; concentration of the conventional bispecific antibody of 3μg / ml; bsc concentration 17-lAxCD 100 ng / ml; E: T relations as indicated. Pigura ti: The DNA and protein sequence of the bscCD19xCD3 antibody (variant containing the iquette-FLAG).

They indicate the numbers of the nucleotide positions (nt), the sequence of the corresponding amino acid is described following the nucleotide sequence. The sequence encoding the DNA for the bispecific antibody starts at position 1 and starts at the 'ic? i 1593. The first six nt (position -10 a - and the last six nt (position 1596 to 1601) contain the restriction sites of enzyme cleavage for EcoRI and SalI, respectively, nucleotides 1 to 57 specify the sequence of the leader nucleotides 82 to 414 and 460 to 831 encode VLCD19 and 19, respectively, nucleotides 847 to 1203 and 580 to 1575 encode VHCD3 and VLCD3, respectively, and nucleotides 1576 to 1593 encode a His tag. depletion of primary CD19 + B cells (malignant) and recruitment of autologous primary T lymphocytes to birds .e bscCD19xCD3 A) Start point (t = 0): n = 3 x 106 PBL / j.ozo where it was seeded in a healthy tissue culture plate -24 in a volume of 1 ml medium RPMl 1640, each 0 one, supplemented with 10% FCS each. The initial percentage of CD19 + B cells is indicated as well as . of CD4 + - and CD8 + T cells. BG) The relative counts of By CD4 + and CD8 + T cells after t = 5 days of incubation at 5 37 ° C / 5% C02 in the absence (BC) or presence (DG) of bscCD19xCD3 (concentrations as indicated) with or without 60 U / ml IL-2. The negative controls contained in the bispecific single chain antibody (17-lAxCD3) with the specificity of the irrelevant target cell or without bispecific antibody (C). Fig. 10: Purification steps for bscCD19xCD3 isolate 11: SDS-PAGE analysis for the purity of bscCD19xCD3. A 4-12% SDS gradient polyacrylamide gel stained with colloidal Coomassie blue is shown. Rows 1 to 6, molecular size binders; row 2, superfluous cell culture; row 3 active fraction by cation exchange chromatography; row 4; active fraction of chelated affinity chromatography of cobalt; row 5, active fraction of the gel filtration. Equal amounts of protein (2 μg) of the superfluous cell culture and various column fractions were analyzed. The size in kDa of the molecular weight standards is indicated at, 'Gap. The arrow shows the position of bscCD19xCD3. . '.gura? 2: Cation exchange chromatography of .scCD19-rCD3. Protein concentration was measured through absorption at 280 nm (mAU, left).

The elution profile of the protein is shown by the solid line. The gradient profile of the NaCl step is shown by the straight solid line (% B, right) and the fractions collected are indicated by the dotted line. The bscCD19xCD3 was detected in fraction F6. 7: Fig. 13: Cobalt chelate affinity chromatography of bscCD19xCD3. The concentration of the protein was measured by absorption at 280 nm (mAU, left) . The elution profile of the protein is shown by the solid line. The imidazole gradient is shown by the straight solid line (% B, right) and the fractions collected are indicated by the dotted line. The bscCD19xCD3 was detected in fraction F7. elgura 14: Gel filtration of ant i -CD19xanti-CD3. Protein concentration was measured through absorption at 280 nm (mAU, left). The profile of - Protein release is shown by the solid line. The dotted lines indicate the fractions collected. The bscCD19xCD3 was found in fraction F7 corresponding to the molecular size of approximately 60 kDa. • 'cure .15: Blood levels of gamma-giutamyl transferase (GGT) in response to treatments with , cCD19xCD3. The GGT levels were determined by a ÜL .. all standard clinical biochemistry and were expressed as unit / 1. The time axis showed days (d) after the start of the first treatment with drugs e, started with zero hours (h) following the additions of the individual drug. The arrows indicate the points "time of drug administration.

Ultrasound measurements of spleen of patient A-B. A: Determination of spleen size dated April 12, 1999, before bscCD19xCD3 therapy. The figure shows the enlarged spleen (size 146 mm x 69.2 mm) that is due to the infiltration of malignant B cells. B: Determination of the size of the spleen dated April 16, 1999 after treatment with 3 μg on April 14, followed by 10 μg on April 15. The sample shows a shrinkage of the spleen at a size of 132 mm x 58.9 mm caused by systemic treatment with bscCD19xCD3. The discrepancies of the simple measures for the size value shown in Table 1 are explained by the determination of the size of the organ based on ultrasound in different spatial planes. The two dimensions are marked by (+) and (x). Figure 17: Ci leukocyte count in blood in response to treatments with bscCD19xCD3. The number of leukocytes is given as Giga parts / liter. The time axis shows < (d) after the start of the first treatment with the drug and starts with zero hours (h) followed by the 0 individual additions of the drug. The arrows indicate the time points of drug administration. '.gura _0: Blood levels of reactive protein C (CRP) in L5 response to treatments with bscCD19xCD3. CRP levels were determined by a standard biochemical standard method and expressed as mg / dL. The time axis shows days (d) after the start of the first treatment with the drug, and starting with zero hours '((li) followed by the individual additions of the drug The arrows indicate the time points of the administration of the drug.' Cure 19: z the blood levels of the necrosis factor alpha .5 I love (TNF) in response to treatments with '' 5cCDi .cCD3. TNF levels were determined by ELISA and expressed as ng / ml. The time axis shows days (d) after the start of the first treatment with the drug and starts with zero hours (h) followed by the individual additions of the drug. The arrows indicate the time points of the administration of the drug. .cura 20: Blood levels of interleukin-6 (IL-6) in response to treatments with bscCD19xCD3. The levels of IL-6 were determined by ELISA and expressed as pg / ml. The time axis shows days 1) after the start of the first treatment with the drug, and starts with zero hours (h) followed by the individual additions of the drug. The arrows affect the time points of the administration of the drug. Figure 21: Blood levels of interleukin-8 (IL-8) in response to treatments with bscCD19xCD3. The levels of IL-8 were determined by ELISA and expressed in pg / ml. The time axis shows days (d) after the start of the first treatment with the drug, starting at zero, hours (h) followed by the individual additions of the drug. The arrows indicate the time points of drug administration, "laura 22: Blood levels of the alpha chain of the soluble heterologous-2 receptor (IL-2R) in response to treatments with bscCD19xCD3. were determined by ELISA and expressed as Units / ml The time axis shows days (d) after the start of the first treatment with the drug, with zero hours (h) followed by the individual additions of the drug. the time points of drug administration The invention will now be described with reference to the following biological examples that are merely illustrative and should not be construed as limiting in the scope of the present invention. ~ Pimplo 1: Cloning of. Variable domains (V) of camunoc obulin The domains of the light chain V (VL) and the heavy chain V (VH) of hybridoma HD37 (22) were cloned according to the standard PCR methods (23). A synthesis of cDNA was carried out with the bases oligo dT and Taq polymerase. _Í ta ca Bases 5 'Ll: GAAGCACGCGTAGATATCKTGMTSACCCAAWCTCCA [SEC. ID NO: 1] 3 'K: GAAGATGGATCCAGCGGCCGCAGCATCAGC [SEC. ID NO: 2] 5 'Hl: CAGCCGGCCATGGCGCAGGTSCAGCTGCAGSAG [SEC. ID NO: 3] • 'G:? .CCAGGGGCCAGTGGATAGACAAGCTTGGGTGTCGTTTT [SEC. ID NO: 4] 'VLB5RRV: AGGTGTACACTCCATATCCAGCTGACCCAGTCTCCA [SEC. ID NO: 5] 3 'VLGS15: GGAGCCGCCGCCGCCAGAACCACCACCTTTGATCTCGAGCTTGGTCCC [SEC. • -j NO:.] 5 'VHGS15: GGCGGCGGCGGCTCCGGTGGTGGTGGTTCTCAGGTSMARCTGCAGSAGTCWGG [SEC. ID NO: 71 3 'VHBspEl: AATCCGGAGGAGACGGTGACCGTGGTCCCTTGGCCCCAG [SEC. ID NO: 8] For the amplification of the V domains through PCR we used the bases 5'Ll and 3'K, flanking the VL domain, and 5'H1 and 3'G for the heavy chain based on the bases described in Dübel et al. (24) The cDNA of the anti-CD3 scFv fragment was kindly roveyed by A. Traunecker (25). .3 emplo 2: Construction of Bispecific Simple Chain Fragments and Eukaryotic Expression To obtain the scFv fragment of the anti-CD19, the VL- and VH- regions were cloned into plasmic.io vectors separately as templates for a specific VL PCR. - and VH- using the base 5 oligonucleotide pairs 5VLB5RRV / 3 'VLGS15 and 5' VHGS15 / 3 'VHBspEl, respectively. Thus, when overlapping the complementary sequences were introduced into the PCR products, which were combined to form the 15-amino acid (Gly4Ser?) 3-i.u linker coding sequence during the subsequent PCR fusion. This step of amplification was carried out with the base pair 'VLB5RRV / 3' VHBspEl and the resulting fusion product i, or rather the scFv fragment anti-CD19) was cleaved with the restriction enzymes EcoRV and BspEl and "- In this way they were cloned into a written KS vector (Estratagen) that contains the coding sequence (EcoRl / SalL - cloned) of the antisense / anti-CD3 bispecific single chain antibody with an N-terminal FLAG tag [1] or the modified version without FLAG / epitope (21), thus replacing anti-17-lA- with the specificity of anti-CD-19 and preserving the 5- amino acid (Gly Ser?) I-linker by connecting the anti-CD3 scFV terminal C fragment, respectively. Subsequently, the DNA fragments encoding the two versions of the anti-CD19 / anti-CD3 single-chain bispecific anti-CD3 antibody with the VLCDI9 domain arrangement "VHCD3-VHCD3-LCD3 was subcloned EcoR / SalL into the described expression vector pEF-DHRF [1], respectively The resulting plasmid DNAs were subjected to CHO deficient DHFR cells through electroporation: selection, gene amplification and protein production were performed as described [1]. Examples are illustrated with the results obtained with the version containing FLAG of bscCD19xCD3. Purification of bscCD19xCD3 from the superfluous of the transfected CHO cells produced 4 mg / liter of superfluous culture. The bsc-Ab was purified through the histidine tail of i.erminal C through affinity chromatography on a Ni-NTA column as described [1]. The bsc-Ab was elucidated from the Ni-Nta column as a distinct peak with a concentration of 200 mM imidazole. The SDS-Page were carried out in accordance with Laemmliu (26) with a ,)F! I believed 12% followed by spotting with bright blue •. 'omassie R250 to analyze the purification of bsc-Ab. The results of the SDS-PAGE analysis (Figure 1) showed the expected size of bsc-Ab (60 kDa). 31! J enripio 3: Agglomeration properties of bscCD19xCD3 The agglomeration specificities of bsc-Ab to CD3 and CD19 were shown through flow cytometric analysis in Jurkat CD3-positive cells, human PBMC and a number of different cell lines of the B cell CD19 -positive including Bli I, SKW6.4, Daudi, BJAB and Raj i. The CD10 positive B cell lines Daudi, Raj i, BJAB (reports Burkitt), SKW 6.4 (human EBV transformed into B cell) and Blin-1 (pre-B cell line) were used in flow cytometric analysis and in the chromium release assays. Jurkat is a CD3-positive T-cell line and the NCI and L363 plasmacytoma cell lines were negative for the two surface molecules, CD3 and CD19. The cell lines were cultured in full RPMl 1640 (Biochrome) with 10% FCS (GIBCO). Cells of 1 x 10s were washed with PBS, resuspended in 200 μl PBS with 10% Vernimmun (Centeon, Marburg, Germany) and 0.1% NaN3 and incubated for 30 minutes at 4 ° C. After a centrifugation step (100 x g, 5 min) cells were incubated in 50 μl of bscCD19xCD3 (200 μg / ml in PBS with 10% Venimmun and 0.1% NaN3) for 30 minutes at 4 ° C. The cells were washed twice with PBS. For the detection of bsc-Ab, a FITC conjugated antibody against the His tag (Dianova) was used. The irrelevant bsc-Ab 17-lAxCD3, produced by the same expression system as in bscCD19xCD3, or the His-tagged antibody only served as negative controls. Flow cytometry was performed with a FacScan from Becton Dickinson. No agglomeration was detected in the BL60 cells, which does not express either CD19 or CD3 (Figure 2). Example 4: Cytotoxic activity of bsc-ABCD19xCD3 against CD19-positive lymphoma cells. The bscCD19xCD3 antibody proved to be highly cytotoxic for several lymphoma cell lines in a 51Cr release assay (Figure 3). Peripheral blood mononuclear cells (PBMC) as effector cells were isolated fresh from random donors using Lymphoprep ™ gradient centrifugation (Nicomed) with subsequent steps of 100 x g centrifugation to remove thrombocytes. CD19-positive cells were reduced using Dinabeads® M-450 CD19 Dynal). The populations of reduced cells were analyzed through flow cytometry (Beckton Dickinson), which showed a 99% depletion of CD19-positive cells. The PBMC were incubated overnight at 37 ° C, 5% C02 CD19-positive B cell lines (Raji, Blin I, Daudi, GJA, SKW6.4) using them as target cells. Cytotoxicity was measured in a standard chromium release assay in well-plates-96 with round bottom (Nunc) using a complete medium RPMl 1640 (Biochrome) with 10% FCS (GIBCO). Unstimulated PBMCs were added in a volume of 80 μl of medium each well containing 20 μl of bsc-Ab in different concentrations. Then 100 μl of 51Cr labeled white cells (1 x 104) were added, the plates were centrifuged for 3 minutes at 100 x g and incubated for 4 hours at 37 ° C, 5% C02. After a further centrifugation step 50 μl superfluous was removed and analyzed for the 51 Cr released in a gamma counter (TopCount, Canberra nackard). Spontaneous release was measured by incubating the target cells without effector cells or antibodies, and maximum release was determined by incubating the target cells with 10% TrtonX-100. Incubation of the target cells with bscAb without effector cells did not I have a measurable lysis. The specific percentage of the lysis was calculated with a specific release (%) = [(cpm, experimental release) - (cpm, spontaneous release)] / [(cpm, maximum release) - (cpm, spontaneous release)] x 100. All tests were carried out in triplicates. SD within the -ijplicados was, in all the experiments, below 6%. To approximate the conditions in vi ve we use PBMC without stimulating healthy donors as effector cells. Rapid induction of cytotoxicity within 4 hours can be observed without any T-cell pre-stimulation protocol. As a control, an anti-bsc antibody with different tumor specificity (bscl7 - 1AXCD3) but generated by the same system as antibody bscCD19xCD3 showed a lysis effect that is not significantly above the environment of the medium. Additionally, no cytotoxic activity could be observed using the NCI and L363 plasmacytoma cell lines that do not express CD19 as target cells (Figure 4). In competition assays, the amounts of increased cytotoxic activity of the CD19-specific parental monoclonal antibody HD37 of bscCD19xCD3 can be almost completely blocked (Figure 5). These controls show that the effects of cytotoxicity mediated by bscCD19xCD3 are antigen-specific. To obtain more information about the molecular mechanism on how the bscCD19xCD3 antibody kills the CD19-positive target cells we have tried to block the cytotoxicity mediated by bscCD19xCD3 through EGTA. As shown in Figure 6, the cytotoxic activity of bscCD19xCD3 can be completely blocked by EGTA indicating that the specific lysis is a T cell mediated effect (probably through the way of performing) instead of a direct effect (for example induction by apoptosis) of the antibody by itself. Using T cells without stimulating even at lower antibody concentrations at 1 ng / ml, a significant cytotoxic effect against Blm-1 cells could be observed (Figure 7). Even at relatively low ratios of E: T (5: 1; 2.5: 1) and at low antibody concentrations of 10-100 pg / ml the bscCD19xCD3 antibody was able to rapidly induce the specific cytotoxic activity of the unstimulated T cell (Figure 7). In contrast, a conventional bispecific antibody CD19xCD3 generated by the hybrid-hibpdoma technique (5-7, 27) did not show significant cytotoxic activity under these conditions even at concentrations up to 3000ng / ml (Figure 7). This conventional bispecific antibody requires further pre-stimulation of the T cell and high antibody concentrations of around 100 ng / ml to induce T cell-specific cytotoxicity (not shown) that is consistent with the literature (5-7). , 27) ', Example 5: Depletion of the iaialignant primary B cells) through autologous T cells through the cytotoxic activity of bscCD19xCD3 In order to evaluate the cytotoxic activity of bscCD19xCD3 in early malignant B cells, peripheral blood monocleotide cells (PBMC) from a patient suffering from B-CLL (chronic lymphatic leukemia derived from B cells) were isolated by density gradient centrifugation Ficoll. These cells were consecutively cultured in the presence or absence of bscCD19xCD3 for 5 days at 37 ° C / 5% C02 in a RPMl 1640 medium supplemented with 10% FCS and, optionally, with 60 U / ml IL-2. Fluctometric analysis revealed that peripheral blood lymphocytes (PBL) of this patient NHL (non-Hodgkin's lymphoma) in particular (which was subsequently treated systemically with bscCD19xCD3, see example 7) contained 92.6% B cells CD19-positive (= target cells) and 7.4% CD3-positive T lymphocytes (= effector cells) at a CD4 / CD8 ratio of 2.6: 4.8. The vast majority of these CD19-positive B cells consisted of malignant cells. 3xl06 PBL / ml per well were seeded in a volume of ml each into a well tissue culture plate 24. Since the negative controls served as culture medium plus IL-2 and the culture medium plus IL-2 with the irrelevant bispecific antibody bccl7 - 1AXCD3 (1) at a concentration of 0.5 μg / ml. As shown in Figure 9, no depletion of CD19 positive cells was detected under these conditions after 5 days of incubation. However, when bscCD19xCD3 was added at concentrations of 0.5 μg / ml or 0.05 μg / ml (in the presence or absence of IL-2) almost all CD19-positive B cells were killed. The culture cells at that time consisted mainly of T lymphocytes with a CD4 / CD8 T cell ratio of approximately 1: 2 to 1: 3. This demonstrated the exceptional cytotoxicity of bscCD19xCD3 towards CD19 positive B cells, since rotal depletion of primary B cells by autologous T cells can be induced at a concentration of only 50 ng / ml at a high initial effector target cell ratio unfavorable less than 1:10, even without IL-2 or other type of additional stimulation of the T cell. Example 6: Purification of bscCD19xCD3 for therapeutic use The bscCD19xCD3 was produced in the ovarian cells of a Chinese hamster stably transfected with a vector expression (pEF-DHFR, see example 2) encoding the bscCD19xCD3 and, additionally, a hexahist idine and a FLAG tag, the cells were grown in a serum-free medium (Tencyte) in a hollow fiber reactor (Unisyn) . Five hundred ml of superfluous cell culture was collected and sterile filtered through a 2 μm filter (AcroCap; Pall Gelman). The bscCD19xCD3 was detected and quantified in a western blotting using anti-FLAG IgG (Sigma) and goat-anti-mouse IgG coupled to an Alkaline Phosphatase (Sigma). Detection was carried out using chemiluminescence using the BCIP / NBT system (Devitron). Protein concentrations were determined by the Bradford assay (Biorad), using bovine IgG (Biorad) as a standard protein. The purity of the column fractions was evaluated by reducing sodium dodecyl sulfate (SDS) Bis / Tris 4-12% polyacrylamide gradient gel electrophoresis (PAGE) using a MOPS buffer system (Novex). The purification of bscCD19xCD3 to homogeneity required a cation exchange chromatography, a coagulation keiato affinity chromatography and, as a final step, gel filtration. These purification steps were carried out using the standard protocols (see below). In Figure 10 a flow diagram of the purification process is shown. Cation exchange chromatography: The superfluous cell culture of the CHO cells was mixed with two volumes of Buffer C (30 mM sulphonic acid of Morgolinoethane [MES], 20 mM NaCl, 3 mM EDTA, 0.3 mM of benzamide hydrochloride, pH 5.5) and passed over a Sepharose Rapid Flow cation exchange column of 70 ml-SP (Pharmacy) at a flow rate of 20 ml / min. The column was equilibrated with a Buffer A, the '? ScCD19xCD3 was elucidated with a gradient step at 45% of Buffer G (20 mM MES, 1 M NaCl, pH 5.8) in Buffer A. The wash received volumes of 0.045. 1 M Tris / HCl, pH 8.5, containing 47 mM of imidazole, and subsequently sterile filtered (0.2 μm; AcroCap). A typical elution profile of the cation exchange chromatography is shown in Figure 12. The bscCD19xCD3 was contained in fraction 6. Cobalt chelate affinity purification: The wash of the cation exchange column was passed at a rate flow rate of 2.5 ml / min on a 10 ml column of Ocellatin Sepharose Rapid Flow (Pharmacy) equilibrated in an AO Buffer (50 mM rMa2HP04, 400 mM NaCl, pH 8.0). The column was pre-equilibrated with a 0.1 M solution of cobalt chloride. After washing with 33 volumes of the AO Buffer column, Buffer A (50 mM Na2HP04, 400 mN NaCl, 2 mM imidazole, pH 6.4) and a 0-12% gradient of Buffer B (50 mM Na2 HP04 , 400 mM 500 mM NaCl imidazole, pH 6.4) in Buffer A, the bscCD19xCD3 was washed in one pass through 30 ml of Buffer B 100%. The wash was sterile filtered followed by approximately a concentration of 10 folds in a MacroSep device (Pall Gelman, 10 kD cut). A typical elution profile for the affinity chromatography of cobalt chelate is shown in Figure 13. The bscCD19xCD3 was detected in fraction No. 7. Gel filtration: The concentrated wash of the cobalt chelate affinity column was loaded at a flow rate of 0.75 ml / min in a 124 ml column of High Load Superdex (Pharmacy, prep grade) equilibrated with a phosphate buffered saline (Gibco) The bscCD19xCD3 washed in a fraction with a molecular size corresponding to approximately 55 kDa (Figure 14, fraction No. 7). The gel filtration fraction containing bscCD19xCD3 was supplemented with 5% human serum albumin (Behring) followed by a ile filtration through a 0.1 μm filter (Millex, Millipore). The abundance of bscCD19xCD3 in a superfluous cell culture and the various fractions of active columns, as analyzed by SDS-GE,. e is shown in Figure 11. The bscCD19xCD3 was the major protein band detected in the superfluous cell culture (row 19). The highly purified anti-CD19xant i-CD3, which was used for human therapy, showed no detectable impurities (Figure 11, row 5). api 7: Clinical use of bscCD19xCD3 in a patient, with the use of cell B In compassionate use a patient (AB, female, born in 1937) who suffers from chronic lymphatic leukemia derived from cell B (B-CLL ) has been treated with a bispecific single-chain antibody. 'and Patient's Soria and Reasoning: The patient was diagnosed with B-CLL in 1992. At the time of initial diagnosis the disease had affected several regions of the lymph nodes and the spleen; additionally, hemolytic anemia of autoimmune origin and a '- immunoglobulin disease. The patient had struma nodosa that is well controlled in eutyreotic condition through treatment with 2.5 mg / d of carbimazole. The patient has received multiple courses of chemotherapy with chlorambucil and prednisone from 1992 to 1994. Following the progress of the disease, treatment has changed to cyclophosphamide, doxorubicin, vmcristma and prednisone (CHOP, 8 cycles) and a remission of more than a year. After a new relapse, the patient received another 6 cycles of CHOP, followed by chlorambucil and prednisone and a single chlorambucil course alone that did not cause any improvement in the disease. In December 1998, irradiation of the spleen was performed to control the splenomegaly of the patient. The patient experienced deep depression of the bone marrow with multiple infectious complications. Their anemia and thrombocytopenia require frequent transfusions of red blood cells and replacement of platelets. Due to the advanced stage of the disease and impaired function of the bone marrow, so aggressive chemotherapy or high doses was not indicated in this patient. The treatment with the anti-CD20 antibody ptuximab was not correct since the efficacy of rituximab in B-CLL was not clearly demonstrated. A FACS analysis revealed that 95% of the patient's peripheral blood cells were jnositive CD19 cells while 77% of the cells expressed the CD20 aitigen. Incubation of the peripheral blood cells of the patient with bscCD19xCD3 showed a pronounced depletion of CD19 positive B cells (see example 5). Therefore, the doctors decided to treat the patient with the innovative bscCD19xCD3 in a compassionate way. The patient was informed in detail about the novel compound and about the potential risks and benefits of this treatment. She fully understood the explanations and gave written informed consent to this compassionate use. Description of Clinical Administration: Prior to initiating treatment, the patient underwent a clinical examination and extensive diagnostic procedures to verify the extent of the disease and to exclude any additional risk factors. The patient was in a good clinical condition with anemia, trobocytopenia and weight loss but without any cardiovascular deficiency or other complications that avoided the use of bscCD19xCD3. During the night before the first days of treatment, the patient suffered from migraine. For the administration of bscCD19xCD3, the patient was kept in hospital custody under intensive care conditions to ensure rapid treatment in case of any emergency that might occur. To avoid any acute reaction to the cytokine and the omplications of tumor lysis, the patient received a prophylactic IV dose of 2 mg of clemasthma (Tavegil®) and 200 mg of cimetidma (Tagamet®) as well as 300 mg of alopurmol and 20 mg of omeprazole (Antra®). Alkalization and heparization was performed through the treatment and the follow-up periods. Additionally, the patient received all the treatment necessary symptomatic. Blood samples were taken before and during the administration of the drug to follow the biochemical, hematological and immunological parameters. ig administration of bscCD19xCD3 (April 14, 1999): o.- The patient received a first dose of 3μg bscCDl !? CD3 as mm-infusion in isotonic phosphate buffer containing 5% human serum albumin (HSA) During the infusion, the patient had no adverse effect. About 1 hour after the? infusion, the patient had chills for about 5 minutes followed by sweating, a moderate drop in blood pressure for a few hours. Additionally, his headache worsened slightly. Ce treated the patient with another 2 mg of Tavegil®, 250 5 mg of prednisolone (Solu-Decort m®) and 50 mg petidma (Dolantm®). All symptoms released without sequelae on the same day. 2nd administration of bscCD19xCD3 (April 15, 1999) A second dose of lOμg bscCD19xCD3 was given a day later under the same conditions. About one hour after the infusion, the patients presented chills, fever (39.2 °), mild hyperventilation and hypotensive reaction. Patients were treated with Tavegil 2 mg, Tagamet 200 mg 200 rng of Solu-Decortin and 15 mg of piritramide (Dipidolor®). For the stabilization of her cardiovascular function, the patient received an infusion of dopamma and received a volume replacement. After this treatment, the symptoms decreased significantly. However, it was transferred to the - Consult the cardiology department at night to ensure correct monitoring of vital signs and immediate intervention in case of emergency. The patient was transferred to the normal guard the following morning without having any additional complication. During the following 3 days, the patient continues to present subfebrile temperature (around 37.2 ° C) and develops minor pleural effusion a day after the second dose (April 16, 1999) and a slight edema in the lower extremities (April 18). , 1999) . Cardiovascular function remained stable and laboratory evaluations revealed no significant changes with respect to safety,. except for an increase of β-glutamyltransferase after < The second dose of bscCD19xCD3 (Figure 15) Since the bscCD19xCD3 was tolerated by the patient and the adverse effects could be controlled with the symptomatic treatment, the administration of the novel bscCD19xCD3 will continue in this patient Clinical and Immunological Ethics of bscCD19xCD3 Results Clinical: An ultrasound examination of the spleen and five abdominal and axillary lymph nodes was performed one day and 4 days after the administration of the second dose of bscCD19xCD3, one day after the dose of lOμg (April 16, 1999). Lymph nodes as well as the spleen already showed a shrinkage of around 20% compared to the baseline evaluation.This observation was confirmed in a second evaluation on April 19, 1999. The weight of the spleen decreased 350 g (from 16309 g in the baseline at 1280 g on April 19, 1999) (Table 1, Figure 16) Hematological Results The number of white blood cells, which include the majority of the Malignant B cells decreased during the course of treatment and on follow-up days (Table 2; figure 17). Reactive protein C (CRP) is an acute phase reaction protein that reflects the activation of the T cell and the effect of pro-inflammatory cytokines. They increased significantly after the administration of 10 μg bscCD19xCD3, followed by a continuous decrease during the next 3 days of observation (Table 2, Figure 18). Immunological Results: The level of cytokines in serum that reflects the acute immunological response to the administration of the compound, was measured before and during several intervals after the administration of the novel compound. The levels ,?? serum of cytokines and soluble IL-2 receptor were measured through a quantitative ELISA study according to the manufacturer's instructions. The tumor necrosis factor TNF-a significantly increased in a dose-dependent manner within the first hour after administration of bscCD19xCD3 (FIG. 19). Interleukin 6 (IL-6) and interleukin 8 (IL-8) also showed a significant increase and an increase in dose dependence. Their maximum levels were observed from 2 to 4 hours after the administration of bscCD19xCD3 (FIGS., twenty-one) . All cytokines returned to baseline levels within a few hours. The soluble receptor IL-2 was already elevated in the baseline, which is explained by the mass of malignant B cells expressing the IL-2 receptor. After administration of the novel bscCD19xCD3, an i-icrement of the IL-2 receptor was observed, indicating an activation of the effector cells (Figure 22). Conclusion: The innovative bscCD19xCD3 was administered safely to a patient suffering from refractory B-CLL. The tolerability of bscCD19xCD3 at doses of 3 μf and 10 'was acceptable and could be well controlled by means of prophylactic measures and symptomatic treatment. The novel bscCD19xCD3 caused a shrinkage of the previously enlarged spleen and lymph nodes of the patient, as shown in the ultrasound examination. Since the lengthening of the spleen and ., two lymphatics is caused by infiltrations of malignant B cells, shrinkage reflects the destruction of malignant B cells as a result of administration of bscCD19xCD3. In sharp contrast to any other CD19xCD3 bispecific antibody known in the art, the bispecific CD19xCD3 antibody of the invention (bscCD19xCD3) exhibits clinical efficacy in the B cell derived from a non-Hodgkin lymphoma which is measured by the shrinkage of the cells. Lymphoid organs infiltrated by malignant cells. Advantageously, bscCD19xCD3 proved to be effective (clinically and surprisingly at low doses which are well tolerated after systemic administration.Thus, the clinical efficacy of bscCD19xCD3 confirms its exceptional cytotoxic activity as determined in vitro Table 1. The effect of bscCD19xCD3 in the size of the lymphatic tissues and the spleen in a patient suffering from L.3 B-cell lymphoma Measurements by Ultrasound 'i ril, 121999 April, 161999 April, 191999 Abdominal lymph nodes 1) 54 x 29 x 14 mm 42x30x 13 mm 42 x 30x 14 mm 2) 56x33x 18 mm 43x33x 18 mm 43 x 30x 16 mm 3) 46 x 32 x 27 mn. 46 x 31 x 22 mm 47 x 32 x 23 mm left axillary 36 24x 16 mm 34x22x 15 mm 30 x 22x 14 mm right 37x24x 13 mm 33x20x 11 mm 32 x 23x 14 mm Spleen 270 x 146x69 mm 265x 132x64 mm 265x 128x63mm 1630 g 1340 g 1280 g The sizes of the three abdominal lymph nodes, one left axillary lymph node and one right and the spleen were determined and measured through sonography using a Toshiba 'SA100 device. The sizes are presented in three dimensions eu mm. Spleen weight was calculated from its dimension and density in ultrasound.

Table 2: Blood levels of markers -selected in response to treatments with • -'JDi: CD3.

Blood levels of gamma-glutamyl transferase (GGT), lactate dehydrogenase (LDH) and C-reactive protein (CRP) were determined by standard clinical biochemical methods and are expressed in Units / ml (GGT), Units / 1 (LDH ) and mg / dl (CRP). The number of leukocytes is expressed in points Giga / 1,. and the number of lymphocytes is presented as a total percentage of leukocytes. Baseline levels on April 14, 1999, before treatment is presented in the first row. The response to 3 μg of bscCD19xCD3 on April 15 (which was administered on April 14) is shown in the second row. The response to a second treatment of lOμg composed on the same day is shown in the third row. The levels of the selected markers in the four days following the drug treatments are presented in the following four rows.

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Sequence Listing (1) GENERAL INFORMATION (i) APPLICANT: (A) NAME: DOERKEN, Bernd (B) STREET: Lyckallee 47 (C) CITY: Berlin (D) STATE: none (E COUNTRY Germany (F) POSTAL CODE ( ZIP): 14055 (A) NAME: RIETHMUELLER, Bernd (B) STREET: Finauer Str. 12 (C) CITY: Munich (D) STATE: none (E) COUNTRY: Germany (F) ZIP CODE (ZIP): 80805 (ii) TITLE OF THE INVENTION: Innovative specific CD19xCD3 polypeptides and the uses thereof. (iii) NUMBER OF SEQUENCES: 10 (iv) LEGIBLE FORM IN COMPUTER (A) TYPE OF MEDIA: Flexible disk (B) COMPUTER: IBM compatible PC (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) SOFTWARE: Patentln Relay # 1.0, Version # 1.30 (EPO) (2) INFORMATION FOR SEC. ID NO: 1: (i) SEQUENCE CHARACTERISTICS (A) LENGTH: 36 base pairs (B) TYPE: nucleic acid (C) FILAMENTATION: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) ) DESCRIPTION: / desc = "oligonucleotide" (iii) HYPOTHETICAL: YES (xi) SEQUENCE DESCRIPTION: SEC. ID NO: 1: GAAGCACGCG TAGATATCLT GMTSACCCAA WCTCCA 36 (2) INFORMATION FOR SEC. ID NO: 2: (i) SEQUENCE CHARACTERISTICS (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) FILAMENTATION: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) ) DESCRIPTION: / desc = "ol igonucleót gone" (iii) HYPOTHETICAL: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: CAGCCGGCCA TGGCGCAGGT SCAGCTGCAG SAG 30 (2) SEQ ID NO: 4 (i) SEQUENCE CHARACTERISTICS (A) LENGTH: 39 base pairs (B) TYPE: nucleic acid (C) FILAMENTATION: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "ol igonucleót gone" (iii) HYPOTHETICAL: YES (xi) SEQUENCE DESCRIPTION: SEC. ID NO: 3 CAGCCGGCCA TGGCGCAGGT SCAGCTGCAG SAG 33 (2) INFORMATION FOR SEC. ID NO: 4: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 39 base pairs (B) TYPE: nucleic acid (C) FILAMENTATION: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "ol igonucleót gone" (iii) HYPOTHETICAL: YES (xi) DESCRIPTION OF THE SEQUENCE: SEC. ID NO: 4: ACCAGGGGCC AGTGGATAGA CAAGCTTGGG TGTCGTTT 39 (2) (2) INFORMATION FOR SEC. ID NO: 4: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 36 base pairs (B) TYPE: nucleic acid (C) FILAMENTATION: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "oligonucleotide" (iii) HYPOTHETICAL: YES (xi) DESCRIPTION OF THE SEQUENCE: SEC. ID NO: 5: AGGTGTACAC TCCATATCCA GCTGACCCAG TCTCCA 36 (2) INFORMATION FOR SEC. ID NO: 6: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 48 base pairs (B) TYPE: nucleic acid (C) FILAMENTATION: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "ol igonucleót gone" (iii) HYPOTHETICAL: YES (xi) DESCRIPTION OF THE SEQUENCE: SEC. ID NO: 6: GGAGCCGCCG CCGCCAGAAC CACCACCTTT GATCTCGAGC TTGGTCCC 48 (2) INFORMATION FOR SEC. ID NO: 7: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 48 base pairs (B) TYPE: nucleic acid (C) FILAMENTATION: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "oligonucleotide" (iii) HYPOTHETICAL: YES (xi) DESCRIPTION OF THE SEQUENCE: SEC. ID NO: 7: GGCGGCGGCG GCTCCGGTGG TGGTGGTTCT CAGGTACTGC AGAGTCGG 48 (i) SEQUENCE CHARACTERISTICS (A) LENGTH: 39 base pairs (B) TYPE: nucleic acid (C) FILAMENTATION: linear (ii) TYPE OF MOLECULE: other nucleic acid ( A) DESCRIPTION: / desc = "oligonucleotide" (iii) HYPOTHETICAL: YES (xi) DESCRIPTION OF THE SEQUENCE: SEC. ID NO: 8: AATCCGGAGG AGACGGTGAC CGTGGTCCCT TGGCCCCAG 39 (2) INFORMATION FOR SEC. ID NO: 9: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 1611 base pairs (B) TYPE: nucleic acid (C) FILAMENTATION: DOUBLE (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (iii) HYPOTHETICAL: NO (xi) CHARACTERISTICS: (A) NAME / KEY: CDS (B) LOCATION: 11.1603 (X) DESCRIPTION OF THE SEQUENCE: SEC. ID NO: 9: GAATTCCACC ATG GGA TGG AGC TCT ATC ATC CTC TTC TTG GTA GCA ACA 4 9 Met Gly Trp Ser Cys l ie l ie Leu Phe Leu Val Wing Thr 1 5 10 GCT ACA GGT GTC CAC TCC GAC TAC AAA GAT GAT GAC GAT AAG GAT ATC 97 Al to Thr Gly Val Hi s Ser Asp Tyr Lys Asp Asp Asp Asp Lys Asp H e 1 5 2 0 2 5 CAG CTG ACC CAG TCT CCA GCT TCT TTG GCT GTG TCT CTA GGG CAG AGG 145 Gln Leu Thr Gln Ser Pro Wing Ser Leu Wing Val Ser Leu Gly Gln Arg 30 35 40 45 GC.'C ACC ATC TCC TGC AAG GCC AGC CAA AGT GTT GAT TAT GAT GGT GAT 193 Wing Thr He Ser Cys Lys Wing Ser Gln Ser Val Asp Tyr Asp Gly Asp 50 55 60 AGT TAT TTG AAC TGG TAC CAA CAG ATT CCA GGA CAG CCA CCC AAA CTC 241 Be Tyr Leu Asn Trp Tyr Gln Gln He Pro Gly Gln By Pro Lys Leu 65 70 75 CTC ATC TAT GAT GCA TCC AAT CTA GTT TCT GGG ATC CCA CCC AGG TTT 289 Leu He Tyr Asp Ala Ser Asn Leu Val Ser Gly He Pro Arg Phe 80 85 90, 'H, T GGC AGT GGG TCT GGG ACA GAC TTC ACC CTC AAC ATC CAT CCT GTG 337 Ser Gly Ser Gly Be Gly Thr Asp Phe Thr Leu Asn He His Pro Val 95 100 105 GAG AAG GTG GAT GCT GCA ACC TAT CAC TGT CAG CAA AGT ACT GAG GAT 385 Glu Lys Val Asp Ala Ala Thr Tyr His Cys Gln Gln Ser Thr Glu Asp 110 115 120 125 (- • CG TGG ACG TTC GGT GGA GGG ACC AAG CTC GAG ATC AAA GGT GGT GGT 433 By Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu He Lys Gly Gly Gly 130 135 140 GGT TCT GGC GGC GGC GGC TCC GGT GGT GGT GGT TCT CAG GTG CAG CTG 481 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu 145 150 155 C? G CAG TCT GGG GCT GAG CTG GTG AGG CCT GGG TCC TCA GTG AAG ATT 529 Gln Gln Ser Gly Wing Glu Leu Val Arg Pro Gly Ser Ser Val Lys He 160 165 170 TCC TGC AAG GCT TCT GGC TAT GCA TTC AGT AGC TAC TGG ATG AAC TGG 577 Ser Cys Lys Wing Ser Gly Tyr Wing Phe Ser Ser Tyr Trp Met Asn Trp 175 180 185 GTG AAG CAG AGG CCT GGA CAG GGT CTT GAG TGG ATT GGA CAG ATT TGG 625 Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp He Gly Gln He Trp 190 195 200 205 CCT GGA GAT GGT GAT ACT AAC TAC AAT GGA AAG TTC AAG GGT AAA GCC 673 By Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe Lys Gly Lys Wing 210 215 220 ACT CTG ACT GCA GAC GAA TCC TCC AGC ACA GCC TAC ATG CAA CTC AGC 721 Thr Leu Thr Wing Asp Glu Being Ser Thr Wing Tyr Met Gln Leu Ser 225 230 235 AGC CTA GCA TCT GAG GAC TCT GCG GTC TAT TTC TGT GCA AGA CGG GAG 769 Ser Leu Wing Ser Glu Asp Ser Wing Val Tyr Phe Cys Wing Arg Arg Glu 240 245 250 ACT ACG ACG GTA GGC CGT TAT TAC TAT GCT ATG GAC TAC TGG GGC CAA 817 Thr Thr Thr Val Gly Arg Tyr Tyr Tyr Wing Met Asp Tyr Trp Gly Gln 255 260 265 GGG ACC ACG GTC ACC GTC TCC TCC GGA GGT GGT GGA TCC GAT ATC AAA 865 Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Asp He Lys 270 275 280 285 CTG CAG CAG TCA GGG GCT GAA CTG GCA AGA CCT GGG GCC TCA GTG AAG 913 Leu Gln Gln Ser Gly Wing Glu Leu Wing Arg By Gly Wing Ser Val Lys 290 295 300 ATG TCC TGC AAG ACT TCT GGC TAC ACC TTT ACT AGG TAC ACG ATG CAC 961 Met Ser Cys Lys Thr Ser Gly Tyr Thr Phe Thr Arg Tyr Thr Met His 305 310 315 TGG GTA AAA CAG AGG CCT GGA CAG GGT CTG GAA TGG ATT GGA TAC ATT 1009 Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp He Gly Tyr He 320 325 330 AAT CCT AGC CGT GGT TAT ACT AAT TAC AAT CAG AAG TTC AAG GAC AAG 1057 Asn Pro As Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe Lys Asp Lys 335 340 345 CJC C ACA TTG ACT ACA GAC AAA TCC TCC AGC ACA GCC TAC ATG CAA CTG 1105 Wing Thr Leu Thr Thr Asp Lys Ser Ser Thr Wing Tyr Met Gln Leu 350 355 360 365 AGC AGC CTG ACA TCT GAG GAC TCT GCA GTC TAT TAC TGT GCA AGA TAT 1153 Ser Ser Leu Thr Ser Glu Asp Ser Wing Val Tyr Tyr Cys Wing Arg Tyr 370 375 380 T? T GAT CAT CAT TAC TGC CTT GAC TAC TGG GGC CAA GGC ACC ACT CTC 1201 Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Thr Glu Thr Thr Leu 385 390 395 ACA GTC TCC TCA GTC GAGA GGA AGG GGA GGT TCT GGT GGA AGGA GGA 1249 Thr Val Be Ser Val Glu Gly Gly Ser Gly Gly Ser Gly Gly Ser 400 405 410 (v T TCA CGT GGA GAC GAC ATT CAG CTG ACC CAG TCT CCA GCA ATC 1297 Gly Ser Gly Gly Val Asp Asp He Gln Leu Thr Gln Ser Pro Wing He 415 420 425 ATG TCT TCT GCA TCT CCA GGG GAG AAG GTC ACC ATG ACC TGC AGA GCC AGT 1345 Met Ser Wing Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Wing Ser? -O 435 440 445 rCA AGT GTA AGT TAC ATG AAC TGG TAC CAG CAG AAG TCA GGC ACC TCC 1393 Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser 450 455 460 CCC AAA AGA TGG ATT TAT GAC ACA TCC AAA GTG GCT TCT GGA GTC CCT 1441 By Lys Arg Trp He Tyr Asp Thr Ser Lys Val Wing Ser Gly Val Pro 465 470 475 T? T CGC TTC AGT GGC AGT GGG TCT GGG ACC TCA TAC TCT CTC ACA ATC 1489 Tyr Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr He 480 485 490 AGC AGC ATG GAG GCT GAA GAT GCT GCC ACT TAT TAC TGC CAA CAG TGG 1537 Be Ser Met Glu Wing Glu Asp Wing Wing Thr Tyr Tyr Cys Gln Gln Trp 495 500 505 AGT AGT AAC CCG CTC ACG TTC GGT GCT GGG ACC AAG CTG GAG CTG AAA 1585 Being Ser Asn Pro Leu Thr Phe Gly Wing Gly Thr Lys Leu Glu Leu Lys 510 515 520 525 CAT CAT CAC CAT CAT CAT TAG TCGAC His His His His His His 530 (2) INFORMATION FOR SEC. ID NO: 10: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 531 amino acids (B) TYPE: amino acids (C) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (X) DESCRIPTION OF SEQUENCE: SEC. ID NO: 10: Met Gly Trp Ser Cys He He Leu Phe Leu Val Wing Thr Wing Thr Gly 1 5 10 15 Val His Cer Asp Tyr Lys Asp Asp Asp Asp Lys Asp He Gln Leu Thr 20 25 30 Gln Ser Pro Wing Ser Leu Wing Val Ser Leu Gly Gln Arg Wing Thr He 35 40 45 Ser Cys Lys Wing Gln Ser Val Asp Tyr Asp Gly Asp Ser Tyr Leu 50 55 60 Asn Trp Tyr Gln Gln He Pro Gly Gln Pro Pro Lys Leu Leu He Tyr 65 70 75 80 ?:? p Wing Being Asn Leu Val Ser Gly He Pro Pro Arg Phe Ser Gly Ser 85 90 95 Gly Ser Gly Thr Asp Phe Thr Leu Asn He His Pro Val Glu Val Val 100 105 110 Asp Wing Wing Thr Tyr His Cys Gln Gln Ser Thr Glu Asp Pro Trp Thr 115 120 125 Phe Gly Gly Gly Thr Lys Leu Glu He Lys Gly Gly Gly Gly Ser Gly 130 135 140 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Gln Ser 145 150 155 160 Gly Ala Glu Leu Val Arg Pro Gly Ser Ser Val Lys He Ser Cys Lys 165 170 175 ? the Ser Gly Tyr Ala Phe Ser Ser Tyr Trp Met Asn Trp Val Lys Gln 180 185 190 Arg Pro Gly Gln Gly Leu Glu Trp He Gly Gln He Trp Pro Gly Asp 195 200 205 Gly Asp Thr Asn Tyr Asn Gly Lys Phe Lys Gly Lys Wing Thr Leu Thr 210 215 220 Wing Asp Glu Being Ser Thr Wing Tyr Met Gln Leu Ser Ser Leu Wing 225 230 235 240 Ser Glu Asp Ser Wing Val Tyr Phe Cys Wing Arg Arg Glu Thr Thr Thr 245 250 255 Val Gly Arg Tyr Tyr Tyr Wing Met Asp Tyr Trp Gly Gln Gly Thr Thr 260 265 270 Val Thr Val Ser Ser Gly Gly Gly Gly Ser Asp He Lys Leu Gln Gln 275 280 285 Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala Ser Val Lys Met Ser Cys 290 295 300 Lys Thr Ser Gly Tyr Thr Phe Thr Arg Tyr Thr Met His Trp Val Lys 305 310 315 320 Gln Arg Pro Gly Gln Gly Leu Glu Trp He Gly Tyr He Asn Pro Ser 325 330 335 Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe Lys Asp Lys Wing Thr Leu 340 345 350 Thr Thr Asp Lys Being Ser Thr Wing Tyr Met Gln Leu Ser Ser Leu 355 360 365 Thr Ser Glu Asp Ser Wing Val Tyr Tyr Cys Wing Arg Tyr Tyr Asp Asp 370 375 380 His Tyr Cys Leu Asp Tyr Trp Gly Glr Gly Thr Thr Leu Thr Val Ser ¡¡5 390 395 400 Ser Val Glu Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly 405 410 415 Gly Val Asp Asp He Gln Leu Thr Gln Ser Pro Wing He Met Being Wing 420 425 430 Ser Pro Gly Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val 435 440 445 Ser Tyr Met Asn Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg 450 455 460 Trp He Tyr Asp Thr Ser Lys Val Wing Ser Gly Val Pro Tyr Arg Phe 465 470 475 480 Being Gly Being Gly Being Gly Thr Being Tyr Being Leu Thr Being Being Met 485 490 495 Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn 500 505 510 Pro Leu Thr Phe Gly Wing Gly Thr Lys Leu Glu Leu Lys His His His 515 520 525 His His His 530

Claims (32)

1. CLAIMS 1. A multifunctional chain polypeptide, "imple" comprising (a) a first domain comprising a binder site of an immunoglobulin chain or an antibody that specifically recognizes the CD19 antigen, and (b) a second domain comprises a site. the binder of an immunoglobulin chain or an antibody that specifically recognizes the CD3 antigen 2. The polypeptide of claim 1, wherein these two domains are connected by a linker polypeptide 3. The polypeptide of any one of claims 1 or 2 wherein the first domain or the second domain replicates or corresponds to a VH and L region of a natural antibody 4. The polypeptide of any one of Claims 1 to 3 wherein the antibody is a monoclonal antibody, synthetic antibody or humanized antibody. The polypeptide of any one of Claims 1 to 4, wherein at least one of the domains is a single chain fragment or the variable of the antibody region. 6. The polypeptide of any one of Claims 1 to 5, wherein the domains are arranged in the order VLCD19-VHCD19-VHCD3-VLCD3. 7. The polypeptide of one of Claims 2 to 6, wherein the linker polypeptide comprises a plurality of glycine, alanine and / or serine residues. 8. The polypeptide of any one of claims 2 to 8, wherein the linker polypeptide comprises from 1 to 5 amino acid residues. 9. The polypeptide of any of Claims 2 to 8, wherein the linker polypeptide comprises from 1 to 5 amino acid residues. 10. The polypeptide of any of the Claims 2 through 9, wherein the linker polypeptide comprises the amino acid sequence Gly Gly Gly Gly Ser. The polypeptide of any one of Claims 1 to 10 wherein the first domain comprises at least one CDR of the VH and V region comprising the encoded amino acid sequence,) r the DNA sequence described in Figure 8 of nucleotides 82 to 414 (VL) and nucleotides 460 to 831 (VH) and / or where the second domain comprises at least one CDR of the VH and VL region comprising the amino acid sequence encoded by the DNA sequence described in Figure 8 from nucleotides 847 to 1203 (V) and nucleotides 1258 to 1575 (VL). 12. The polypeptide of any of the Claims 1 to 11, wherein (a) the binding site of the first domain has an affinity of at least about 10 7 M; and / or (b) the binding site of the second domain has an affinity of at least about 10 7M. 13. The polypeptide of any of the eivmdications from 1 to 12, which is a bispecific single-chain antibody. 14. The polypeptide of one of the Claims 1 through 13, comprising at least one additional domain. 15. The polypeptide of Claim 14, wherein the additional domain is linked by covalent or non-covalent linkages. 16. The polypeptide of Claim 14 or Claim 15, wherein at least one additional domain comprises an effector molecule having a conformation suitable for biological activity, capable of sequestering an ion or selective agglomeration to a solid support. or to a determinant, r-selected. 17. A polynucleotide that before expression encodes a polypeptide of any one of Claims 1 to 16. 18. A vector comprising a polynucleotide of Claim 17. 19. A cell transfected with the polynucleotide of Claim 17 or of the vector of Claim 18. 20. A method for the preparation of the polypeptide of any one of Claims 1 to 16, the method of which comprises the anabolic culture of Claim 19 and isolating the polypeptide from the culture. 21. A composition comprising the polypeptide of any one of Claims 1 to 16, the polynucleotide of Claim 17 or the vector of Claim 18. 22. The composition of Claim 21, which is an optionally additional diagnostic composition comprising elements suitable for detection. 23. The composition of Claim 21, which is a diagnostic composition optionally further comprises elements suitable for detections. 24. Use of the polypeptide of any one of Claims 1 to 16, the polynucleotide of Claim 17 or the vector of Claim 18 for the preparation of a pharmaceutical composition for the treatment of B cell malignancies, autoimmune diseases mediated by cell B or depletion of B cells. 25. The use of Claim 24, wherein the malignancy of B cell is not a Hodgkin lymphoma. 26. The use of the polynucleotide of Claim 17 or of the vector of Claim 18 for the preparation of the compositions for therapy of the gene. 27. A method for identifying activators or inhibitors of T cell activation or stimulation comprises (a) culturing T cells and CD19 positive cells, preferably B cells, in the presence of the polypeptide of any one of the Claims of 1 to 16 and optionally in the presence of a component capable of providing a detectable signal in response to activation of the T cell with a compound that will be filtered under conditions that allow T cell activation, and (b) detect the presence or absence of the signal generated from the interaction of the compound with the cells. 28. A method for the production of a pharmaceutical composition comprising the steps of the method of Claim 17 and the formulation of the compound identified in step (b) in a pharmaceutically acceptable form. 29. The method according to Claim 28 or Claim 29, wherein the compound identified in step (b) is modified by replicas of the peptide. 30. A method for the treatment of B cell malignancies, autoimmune diseases mediated by the B cell or the depletion of cells comprising the introduction of the polypeptide of any one of Claims 1 to 16, the polynucleotide of Claim 17 or the vector of Claim 18 in a mammal affected by malignancy or by disease. 31. A method for delaying a pathological condition that is caused by disorders of the B cell, comprising the introduction of the polypeptide of any of Claims 1 to 16, J r > lyucleotide of Claim 17 or the vector of Claim 18 in a mammal affected by the pathological condition. 32. The method of Claim 30 or Claim 31, wherein the mammal is human.