MXPA01000325A - Immunological reagent specifically interacting with the extracellular domain of the human zeta chain - Google Patents

Abstract

The present invention relates to a nucleic acid molecule comprising a nucleic acid sequence encoding at least one complementary determining region (CDR) of a variable region of an antibody, said antibody specifically interacting with the extracellular domain of the human zeta-chain, said antibody being obtainable by immunizing a rat with Jurkat cells and subsequently with a conjugate comprising a carrier molecule and a peptide comprising the 11N-terminal amino acids of the rat zeta-chain. Preferably, the (poly)peptide encoded by the nucleic acid molecule of the invention is a monospecific or bispecific antibody. The invention also relates to pharmaceutical compositions comprising i.a. the nucleic acid molecule or antibody of the invention as well as to kits comprising the aforementioned compounds. Finally, the invention relates to a method for the determination of zeta-chain or eta-chain expression on NK-cells, T-cells or precursors thereof employing the antibody of the invention.

Description

REAGENT IN UNOLOGICAL INTERACTING SPECIFICALLY WITH THE EXTRACELLULAR DOMAIN OF THE HUMAN ZETA CHAIN The present invention relates to a nucleic acid molecule comprising a nucleic acid sequence encoding at least one complementary determining region (CDR) of a variable region of an antibody; said antibody specifically interacting with the extracellular domain of the human zeta chain on the surface of intact cells; being This antibody can be obtained by immunizing a strand with Jurkat cells and subsequently with a conjugate comprising a carrier molecule and a peptide comprising the eleven N-terminal amino acids of the rat zeta chain. Preferably the (poly) peptide encoded by the nucleic acid molecule of the The invention is a monospecific or bispecific antibody. The invention also relates to pharmaceutical compositions comprising, inter alia, the nucleic acid molecule or the antibody of the invention, as well as the equipment comprising the compounds mentioned above. Finally, the invention is refers to a method for determining the expression of zeta chain or eta chain in NK cells, T cells or their precursors, using the antibody of the invention. The zeta chain is part of a family of structurally and functionally related signal transduction molecules, which further comprises the eta chain (one form alternatively folding of the zeta chain) and the high affinity IgE-Fc gamma chain, FceRI. The common characteristics within this family of transmembrane proteins are their long intracellular domain, comprising one or several ITAM 5 sequence motifs (activation motive based on tyrosine, immunoreceptor, zeta-3, eta: 2, gamma: 1), as well as extremely short extracellular domains, consisting of 9 (zeta sequence, identical eta) amino acids or 4 (FceRI-gamma) amino acids. The sequence of the extracellular domains of these proteins is conserved in 100 percent between mouse, rat and man, and very likely also in other species. The zeta chain is expressed as a homodimer or as a heterodimer with the eta chain in T lymphocytes, natural killer (NK) cells and, to some extent, its precursors, exclusively. On the surface of mature T lymphocytes, the zeta chain is structurally and functionally associated with the T cell receptor (TCR) and the CD3 complex. The signals induced by the TCR coupling are transduced to the cytoplasm of the T cell through CD3 and the zeta chain, providing the three ITAM of The zeta chain accounts for most of the signal amplification effect, compared to the only ITAM in the epsilon, delta and gamma chain (different from FceRI-gamma), which constitutes the CD3 complex. On the surface of NK cells, the zeta chain shows a similar association with the IgG-Fc receptor (Fc? RIIIA). When the ? ^^^^^ gl ^? ^ ßÍsáíii ^^^^^^^^. ^^^ - ^^^ --------- ^^^^ - ^^^^^^^ g ^ ^^^^ and | - ^ k; ^^. ..fc? i. t-i i, target cells, coated with antibody, are recognized by NK cells through Fc? RIIIA, the resulting signal is transduced into the cytoplasm through the zeta chain and / or the gamma chain, thereby activating the NK cell that, consequently, performs the lysis of the target cell that was recognized (ADCC: cellular cytotoxicity that depends on the antibody). The TCR complex in mature T lymphocytes, in such a way, is an oligomeric structure, composed of multiple chains (TCR-a / β or TCR-α / d associated with CD3e, CD3d, CD3? And the zeta chain or its product of splice different eta) (Keegan, Immunology focay, 13 (1992) 63-68). Antigenic recognition is obtained by the TCR-a / β or TCR-α / d heterodimers that are devoid of the intracytoplasmic signal transduction domains. The invariable CD3 proteins (heterodimers? / Eyd / e) and the zeta or eta chain (zeta homodimers or zeta-eta heterodimers) are necessary for the correct assembly, transport and efficient expression on the cell surface of the TCR complex whole and transduce the TCR signals (Clevers, Annu., Rev. Immunol., 6 (1988), 629-662; Ashwell, Annu. Rev. Immunol., 8 (1990) 139-167). The signaling requires a conserved 18 amino acid sequence (Reth, Nature, 338 (1989) 383-384; Samelson, J. Biol. Chem., 267 (1992) 24913-24916 (referred to as the tyrosine-based activation motif, of the immunoreceptor (ITAM), which is found three times in the zeta chain, twice in the eta chain and once in each of the CD3 subunits (gamma, delta and epsilon) Each ITAM contains a pair of tyrosine-XX-leucine motifs / isoleucine (YXXL / l), which are separated by 10 or 11 amino acids (Cambier, Immunology Today 16 (1995) 110). The tyrosine residues in each ITAM are rapidly phosphorylated after TCR ligation and serve as anchoring sites for note proleins that can bind phosphotyrosine residues by src s (SH2) homology domains (Cooke, Cell 65 (1991) 281-291; Glaischenhaus, Cell 64 (1991) 511-520, Samelson, Proc. Nati. Acad. Sci USA 87 (1990) 4358-4362; Straus, Cell 70 (1992) 585-5093, Songyang, Cell 72 (1993 767-778; ngyang, Mol. Cell. Biol., 14 (1994) 2777-2785; Isakov, J. Exp. Med., 181 (1995) 375-380). The need for the zeta chain in TCR-mediated signaling was demonstrated by studies of a mutant, zeta-deficient derivative of a T-cell hybridoma specific for the antigen; the mutant line was unable to respond to the antigen and was only poorly responsible to the anti-CD3 antibodies (Sussman, Cell 52 (1988) 85-95). Although the retention of some activity in response to the stimulus of anti-CD3 antibody suggested that the other chains of the TCR complex were able to compensate for the absence of zeta, the studies in which it was demonstrated that the mutant line reacquires the ability to recognize antigen or responding to anti-CD3 antibodies when reconstituted with zeta chain by transfection, clearly document an important role for the zeta chain in signal transduction (Weissman, EMBO J., 8 (1989) 3651-3656). Due to its unique configuration, the chain _. -_ *. *. + * 'j? _? __ ...., zeta can function as the predominant TCR signaling structure and its tripled ITAMs can serve primarily to facilitate TCR signal amplification. The TCR complex in general, and the zeta chain mediated signal transduction, in particular, are involved in both the activation and programmed cell death (apoptosis) of mature T lymphocytes. Experiments in which the zeta chain cytoplasmic tail was bound to unrelated receptors, showed that the cell lines expressing these chimeric receptors could respond to the interlacing of antibody with IL-2, the release and up-regulation of other activation parameters (Irving, Cell 64 (1991) 891-901). Additionally, it was demonstrated that cytotoxic T lymphocytes (CTL) expressing chimeric zeta chain derivatives effect specific lysis of target cells carrying surface molecules recognized by the chimeric zeta receptor (Romeo, Cell 64 (1991) 1037-1046; Romeo, Cell 68 (1992) 889-897). However, this proved to be true only in the case of activated T cells, since inactive T lymphocytes expressing chimeric zeta chain molecules could not be activated nor showed any cytotoxicity against target cells, when stimulated by these cells. chimeric receptors (Brocker, J. Exp. Med., 181 (1995) 1653-1659), which according to biochemical analyzes are not associated with endogenous TCR subunits and, therefore, act as physically independent signaling molecules (Shinkai, Immunity 2 (1995), 401-411). These data thus indicate that the chimeric zeta chain derivatives can be substituted only with the complete TCR complex, in the activated T cells, but not in the inactive ones. TCR 5 mediated apoptosis of mature lymphocytes can be induced if strong TCR reattachment occurs when cells are activated and proliferating (Lenardo, Nature 353 (1991) 858-861, Russell, Proc. Nati, Acad. Sci. USA 88 (1991) 2151-2157; Critchfield, Cell Immunol., 160 (1995) 71-78). The death of mature T cells plays a role critical in peripheral immunological homeostasis and in tolerance. Recent experiments have shown that the zeta chain is required for the efficient induction of T cell apoptosis, by coupling the TCR; and that the CD3 signaling domains only have a minor effect (CD3e) or no effect (CD3? And d) on TCR-mediated apoptosis (Combadiére, J. Exp. Med. 183 (1996) 2109-2117). Additionally, the three zeta chain ITAMs contribute differently to the induction of T-cell apoptosis, the one closest to the N-terminal having the predominant effect, followed by the C-terminal ITAM, which resembles the low contribution of CD3e, and being in the middle completely incapable of inducing apoptosis. T cell development occurs primarily in the thymus, where T cell precursors immigrate from the fetal liver or from the adult bone marrow. When you immigrate to the thymus, you are early progenitor T cells are triplemenle negative (TN: TCR-, CD4", CD8") (Shortman, Annu, Rev. Immunol., 14 (1996) 29-47), but they already express the zeta chain and the CD3 chains (Wíest, J. Exp. Med.180 (1994 1375-1382; Wilson, Int. Immunol., 7 (1995) 1650-1664). In the inductive environment of the thymus, they pass through a series of stages of development before their differentiation to the CD4 + CD8 + double positive thymocytes (DP) (Godfrey, Immunol. Today, 14 (1993) 547-553). The most immature thymocytes CD44 + CD25".- TN and the thymocytes CD44 + CD25 + -TN, derived from them, still show the germline of the TCR genes." The TN-thymocytes of the next stage of maturation, characterized by the superficial phenotype CD44"° CD25 + , however, begin to rearrange the TCRß site. Up to this stage, the zeta chain, although expressed, is probably not necessary for the maturation of the thymocyte (Crompton, Eur. J. Immunol., 24 (1994) 1903-1907). The next step of maturation of TN thymocytes is characterized by the phenotype change from CD44"l0CD25 + to CD44 CD25"; however, it is blocked in the absence of the zeta chain (Crompton, Eur. J. Immunol., 24 (1994) 1903-1907) and requires rearrangement and expression of the TCRß chain (Kishi, EMBO J. 10 (1991) 93 -100, Mombaerts, Nature 360 (1992) 225-231, Shinkai, Science 259 (1993) 822-825). The TCRß chain is associated with an invariant chain called pre-Ta (pTa) (Groettrup, Cell 75 (1993) 283-294), which replaces the TCRa chain still without rearrangement, to form the pre-TCR, probably associated with the chain zeta and the CD3 chains (Van Oers, J. Exp. Med., 182 (1995) 1585-1590). As a consequence of fftH- ^ ¿.-i. Jfr ft £ * - ** .- -tiawt «» * »* *« • »* ----- í- ----- a ---- .-., ..-«. «^ -« - igAJ- ^ M * »Ü-te ^ fe-aw S. the signals mediated by the pre-TCR, the thymocytes advance in their development to the DP stage. At this stage, the thymocytes initiate the rearrangement of their TCRa genes, stop expressing pTa and begin to express low levels of TCR complexes, which resemble the multiple chain composition of the TCR complex in mature T lymphocytes (Von Boehmer, Ann, NY Acad. Sci, 766 (1995) 52-61, Robey, Annu, Rev. Immunol., 12 (1994) 265-705). Since the development of CD44 CD25-TN thymocytes and subsequently that of DP thymocytes is inhibited in the absence of the zeta chain, and can be restored by the expression of a mutant, signaling-deficient zeta chain, without functional ITAM, respectively (Shores , J. Immunol., 159 (1997) 220-230, Shores, Science 266 (1994) 1047-1050), the importance of the zeta chain related to the promotion of surface expression of pre-TCR may exceed that related to its signaling potential. DP thymocytes are further subjected to selection based on the specificity of their TCRs. Thymocytes that express TCR with negligible specificity for the auto-MHC molecules, regardless of which peptide is bound by the MHC protein, die within the thymus, presumably because they can not receive survival signals through their TCR (Robey, Annu Rev. Immunol., 12 (1994) 265-705, Jameson, Annu, Rev. Immunol., 13 (1995) 93-126). In contrast, thymocytes that express TCR with appropriate ligand specificities, ... i. _..__._? ------ -i -i, .--- * - «. . -. ---- ..... - - .-- • - • --- - -----.---- • - - -.--- > • -------- * - - ~ 1 ** ~ - ^? ^ < «*« «* SjÉUfe survive and mature to CD4 + T (CD4 +) or CD8 + cells - monopositive, which show high level of TCR expression. However, thymocytes expressing autoreactive or potentially self-reactive TCRs are eliminated (Robey, Annu, Rev. Immunol., 12 5 (1994) 265-705, Jameson, Annu, Rev. Immunol., 13 (1995) 93-126). . Thus, the coupling of TCR in DP thymolocytes leads to two dramatically different cell futures: survival and subsequent maturation (positive selection) or death by apoptosis (negative selection). 10 While zeta chain ITAMs are not essential for development (Shores, Science 266 (1994) 1047-1050) and selection restricted by MHC (Simpson, Int. Immunol., 1 (1995) 287-293). T SP mature in the thymus, seem to contribute to the formation of the T cell repertoire, amplifying the response of signaling generated by the TCR coupling during the selection of the thymocyte. In fact, the results of a recent study revealed that, although no individual ITAM was specifically required, there was a direct relationship between the number of ITAMs of the zeta chain, within the TCR complex, and the efficiency of positive selection and negative selection (Shores, J. Exp. Med., 185 (1997) 893-900). These results could be expected if the positive and negative selection is dictated primarily by the minimum levels of TCR signaling, and if the magnitude of the signaling response to ligands of different affinity, Critique to determine the future of developing thymocytes is more or less amplified by the triplicate ITAMs of the natural zeta chain, or by a reduced number of ITAMs in the zeta chain mutants, respectively. 5 Thus, it is expected that the repertoire of positively selected thymocytes, in the presence of the natural zeta chain, will differ markedly from those selected in the presence of a zeta chain derivative, deficient in signaling, and that the latter repertoire contains T cells that , otherwise, they would have were negatively selected (Shores, Current Opinion in Immunology, 9 (1997) 380-389). NK cells are large granulated lymphocytes that make up 10 to 15% of peripheral blood lymphocytes (PBL). They are capable of killing tumor cells and certain infected cells Virally, unrestricted by the major histocompatibility complex (MHC) (Trinchíeri, Adv. Immunol., 47 (1989) 187-376, Ritz, Adv. Immunol., (1988) 181-211). Effector cytolytic function does not require prior sensitization or presentation of antigen by accessory cells. These properties allow NK cells to perform an innate host defense before unleashing a specific immune response to the antigen. It is known that NK cells perform two forms of cytolytic activity: natural cytotoxicity and ADCC (antibody-dependent cellular cytotoxicity) which also kills target cells, resistant to natural cytotoxicity.

In contrast to the cytotoxic activity of T cells that is triggered by an activation signal, generated by the coupling of clonotypic TCR, the natural cytotoxicity of NK cells is regulated primarily by inhibitory signals (Yokoyama, J. Exp. Med. , 186 (1997) 1803-1808). The coupling of inhibitory NK cell receptors, by specific binding to MHC class I molecules in the target cells, leads to the inhibition of natural cytotoxicity, which is released in the absence of the MHC class I expression in the target cell, thereby allowing the activation of natural death. NK cell receptors contain inhibitory motifs based on tyrosine, immunoreceptors, intracytoplasmic, (ITIM, consensus sequence l / V- XYXXL) that mediate the inhibitory signals after tyrosine phosphorylation, induced receptor coupling (Muta, Nature , 368 (1994) 70-73, Thomas, J. Exp. Med., 181 (1995) 1953-1956). ADCC is mediated by the Fe Fc? RIIIA receptor of low affinity IgG, and can be demonstrated in vivo by incubating NK cells with target cells coated with antibody. Ligand binding and crosslinking of FcγRIIIA induces NK cell activation, resulting in cytolytic activity, upregulation of surface activation molecules and cytokine secretion (Chehimi, J. Exp. Med. , 75 (1992) 789; Ravetch, Annu, Rev. immunol., 9 (1991) 457). Fc? RIIIA is expressed as a complex comprising the receptor-binding CD16 glycoprotein of the ligand of transmembrane, and two chains that extend in the membrane, gamma and zeta, which are responsible for the assembly of receptor and signal transduction (Anderson, Proc Nati Acad Sci USA, 87 (1990) 2274-2278; Ravetch, Annu, Rev. Immunol., 9 (1991) 457). The gamma chain was originally identified as a high affinity receptor subunit for IgE and belongs to the same family of signal transduction molecules, along with the zeta chain and the eta chain. By coupling FcγRIIIA to the tyrosine of the NK cells, phosphorylation occurs within the ITAMs of the zeta 0 chain and the gamma chain, thereby inducing additional signal transduction events, which include the recruitment of the domain (SH ) 2 of specific Src homology containing proteins, to the Fc? RIIIA complex. As demonstrated by a recent study, NK cell activation mediated by FcγRIIIA appears to be mimicked by the coupling of chimeric zeta chain receptors, transfected into NK cells, thus resembling analog approaches in T cells (Tran, J Immunol., 155 (1995) 100-1009). While it is evident from the foregoing that antibodies that interacted specifically with, and that recognized the extracellular domain of the human zeta chain, on the surface of intact cells, were in high demand for a variety of purposes, such as artificial transduction of signal in T cells or in NK cells, for example, in the treatment of tumors, to date no satisfactory experiments had been reported. The lack of success in the production of a antibody that met that need, may be primarily due to the rather short length (9 amino acids) of this domain, possibly together with the association of the zeta chain with the T cell receptor and the CD3 complex in T cells, or with the IgG-Fc receptor in NK cells. The technical problem underlying the present invention, therefore, was to provide a tool that could be successfully applied to the need identified above. The solution to this technical problem was achieved by providing the modalities characterized in the claims. Accordingly, the present invention relates to a nucleic acid molecule comprising a nucleic acid sequence encoding at least one complementary determinant region (CDR) of a variable region of an antibody; specifically interacting the antibody with the extracellular domain of the human zeta chain on the surface of intact cells; said antibody being obtained by immunization of a rat with Jurkat cells and subsequently with a conjugate comprising a carrier molecule and a peptide comprising the eleven N-terminal amino acids of a rat zeta chain. Said intact cells are preferably T cells or NK cells or their precursors, but they are also artificially transfected cells. In accordance with the present invention, the antibody comprising said CDR is obtainable by sensitizing rats with Jurkat cells (ATCC TIB152) and potentiating them with a conjugate comprising a carrier molecule and the aforementioned peptide. It is also contemplated that alternative immunization strategies may be successful, such as injecting, for example, rats with one or more doses of the conjugate. Although KLH has been used as a carrier when generating the antibody, different carriers could be used successfully. An example of a different carrier is BSA. Since zeta chain molecules occur on the surface of T cells and NK cells, either in association with the TCR or with CD3 or with Fc? RIIIA, or possibly as freely floating homodimers or heterodimers, such interaction can be with either of those forms of zeta or with all of them. Similarly, the antibody can interact with a single zeta chain or specifically with the dimeric structure. Since eta has the same extracellular domain in humans as zeta, the antibody of the invention also recognizes eta in the same conformation and / or in the same associations as the zeta chain. Additionally, the antibody can also specifically interact with the extracellular domain of rat and mouse zeta / eta chains. A particularly advantageous property of the antibody of the invention is the fact that it recognizes zeta / eta in both T cells and NK cells, as well as its precursors. In view of what was known about the structure and associations of the zeta chain, the development of said antibody has been considered really as surprising. The epitopes in the extremely short peptide, of 9 amino acids, are necessarily located near the cell membrane. The intimate and, therefore, intimate spatial association with multimolecular protein complexes on the surface of the cell makes it seem likely that these epitopes are also sterically inaccessible for large structures such as an antibody. Additionally, due to the association of the zeta chain with different multimolecular protein complexes in T lymphocytes and in NK cells, the extracellular zeta chain epitopes, unexpectedly accessible for antibody molecules in T cells, are unlikely to be identical to those of NK cells. In addition, due to sequence identity in humans, rats and mice, the extracellular zeta chain domain represents a self-antigen in the three species, thus making it improbable to obtain antibodies against them, by immunization of mice and rats. The previous notion that the development of this antibody should be considered as extremely surprising, is corroborated by the fact that the most promising approach to obtain Said antibody, failed. Specifically, a bank of combinatorial antibodies, cloned from the RNA of spleen cells of mice immunized with the peptide-KLH conjugate, was extended on the filamentous phage and selected in vitro by alternating scanning in the peptide-BSA conjugate, on CD8 + -T-lymphocytes and NK-purified cells. While enriching the clones of to JíJmtk. * Phage exhibiting Fab-antibody fragments, reactive with the peptide-BSA conjugate was obtained during the last step of scanning in CD8 + T-lymphocytes, most of them were lost during the subsequent scanning step in purified NK cells, which indicates the lack of antibodies within the repertoire that recognize an epitope of extracellular zeta chain in the T lymphocytes and in the NK cells. This was confirmed by testing a large number of clones that were reactive with the peptide-BSA conjugate, of which none could be identified with mixed reactive binding activity. in T lymphocytes and NK cells. Only when the inventors applied the old and rather laborious approach to the generation of said antibody did they eventually succeed. This approach comprised the following steps: A peptide was synthesized that comprised the first eleven N-terminal amino acids of the zeta chain, and they were coupled to KLH by the SH group on cysteine 11. Immunized previously immunized rats with Jurkat cells and mice, with this conjugate, respectively, and selected the hybridoma cell lines obtained against another conjugate consisting of the peptide of 11 amino acids, coupled to BSA. One hundred fifty murine hybridoma cell lines and 45 rat hybridoma cell lines were obtained, which recognized the peptide-BSA conjugate, of which only a rat IgM antibody exhibiting binding activity could be identified. to both T lymphocytes and NK cells, as determined by flow cytometry. The antibodies of the present invention or their corresponding immunoglobulin chain (s9) can be further modified using conventional techniques known in the art, for example, using omission (s), insertion (s), substitution (s), addition ( es) and / or amino acid recombination (s) and / or which is (s) other known modification (s) in this field, either alone or in combination The methods for introducing said modifications into the DNA sequence in which the amino acid sequence of an immunoglobulin chain rests, are well known to those skilled in the art, see, for example, Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory (1989), NY The antibody also may be a chimeric antibody, since it was well known in the art that recognition of an epitope is often dictated by a single CDR, preferably the CDR of the heavy chain, it is contemplated that a CDR of the antibody obtainable according to the program indicated above, will be sufficient to contribute to at least one weak, but significant binding. This is true, preferably, for the antibody that was actually obtained by the strategy referred to above. However, it is preferred that the nucleic acid molecule comprises a nucleic acid sequence encoding at least two CDRs of said variable region.

^^ J ^^^ & amp; amp; > $ & amp; amp; amp; amp; amp; amp; amp; amp; amp; amp; amp; amp; amp; amp; amp; amp; amp; amp; amp; amp; amp; amp; amp; amp; amp; amp; amp; amp; amp; amp; amp; amp; amp; amp; Other nucleic acid molecule encoding three CDRs of said variable region The invention is characterized in that the nucleic acid sequence encodes a VH-chain. In another preferred embodiment of the nucleic acid molecule of the invention, the nucleic acid sequence encodes a VL chain. The nucleic acid molecule of the invention, for example, can be an RNA molecule or a DNA molecule.

In a further preferred embodiment, the nucleic acid molecule of the invention is a DNA molecule. A synthetic or semisynthetic DNA molecule is particularly preferred. In another preferred embodiment of the nucleic acid molecule of the invention, the CDR has one of the following nucleotide sequences: (a) SEQ ID No. 1 (b) SEQ ID No. 3 (c) SEQ ID No. 5 (d) SEQ ID No. 7 (e) SEQ ID No. 9 (f) SEQ ID No. 11. In a particularly preferred embodiment of the ^ n r_i.?AÍ_-_. . -tew »O.Á iíl? nucleic acid molecule of the invention, said VH chain has the nucleotide sequence of SEQ ID No. 13 or encodes the amino acid sequence of SEQ ID No. 14. In a particularly preferred embodiment of the nucleic acid molecule of the invention, said VH chain has the nucleotide sequence of SEQ ID No. 15 or encodes the amino acid sequence of SEQ ID No. 16. The invention also relates to the nucleic acid molecule of any of claims 1 to 6, wherein the CDR encodes one of the amino acid sequences of SEQ ID No. 2, 4, 6, 8, 10 or 12. The invention also relates to a vector comprising the nucleic acid molecule of the invention. The vector of the invention may comprise additional genes, such as marker genes that allow the selection of said vector in a suitable host cell and under suitable conditions. It is preferred that the polynucleotide of the invention be operably linked to expression control sequences that allow the expression of prokaryotic or eukaryotic cells. Expression of the polynucleotide comprises transcription of the polynucleotide into a translatable mRNA. Regulatory elements that ensure expression in eukaryotic cells, preferably mammalian cells, are well known to those skilled in the art. Habitually they comprise regulatory sequences that guarantee the start of transcription and, optionally, poly-A signals that guarantee the termination of transcription and the stabilization of the transcript. Additional regulatory elements may include transcription enhancers as well as translational and / or naturally associated heterologous promoter regions. In this regard, those skilled in the art will readily appreciate that polynucleotides that encode at least the variable domain of the light chain and / or heavy chain can encode the variable domains of both immunoglobulin chains, or only one. Likewise, the polynucleotides can be under the control of the same promoter or can be controlled separately for their expression. Possible regulatory elements that allow expression in prokaryotic host cells comprise, for example, the PL, lac, trp or tac promoter in E. coli, and examples of regulatory elements that allow expression in eukaryotic host cells are the AOX1 promoter. or GAL1, in yeast, or the CMV promoter, SV40, RSV (Rous sarcoma virus), the CMV enhancer, the SV40 enhancer or a globin intron in mammalian cells and other animal cells. In addition to the elements that are responsible for the initiation of transcription, said regulatory elements may also comprise transcription termination signals, such as the SV40-poly-A site or the tk-poly-A site, downstream of the polynucleotide. Additionally, depending on the system - '- "*» "- *' - < "" ': Expression used, it is possible to add to the coding sequence of the polynucleotide of the invention leader sequences, capable of directing the polypeptide to a cellular compartment or of secreting it into the medium, which are well known in the art. The sequence or leader sequences are assembled in appropriate phase with translational initiation and termination sequences and, preferably, a leader sequence capable of directing the secretion of translated protein or a portion of it into the periplasmic space or the extracellular environment. Optionally, the heterologous sequence can encode a fusion protein that includes a C or N terminal identification peptide, which imparts the desired characteristics, for example, stabilization or simplified purification of the expressed recombinant product. In this context, suitable expression vectors are known in the art, such as the cDNA expression vector of Okayama-Berg cDNA (Pharmacia, pCDM8, pRc / CMV, pcDNA, pcDNA3 (In-Vitrogene) or pSPORTI (GIBCO BRL It is preferable that the expression control sequences be eukaryotic promoter systems in vectors capable of transforming or transfecting the eukaryotic host cells, but control sequences for prokaryotic hosts can also be used, once the vector has been incorporated into the host appropriate, the host is maintained under conditions suitable for high-level expression of the nucleotide sequences and, when desired, the collection and purification of immunoglobulin light chains, heavy chains, light / heavy chain dimers or intact antibodies, they can follow the binding fragments or other forms of immunoglobulin, see Beychok, Cells of Immunoglobulin Synthesis, Acade Mec Press N. Y. (1979). The vector of the present invention which, for example, can be a plasmid, a cosmid, a virus or a bacteriophage, is preferably an expression vector. Expression vectors derived from viruses such as retroviruses, vaccinia viruses, adeno-associated viruses, herpes viruses or bovine papilloma virus can be used to deliver the polynucleotides or the vector of the invention to the population of target cells. Methods that are well known to those skilled in the art can be used to construct the recombinant viral 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 in Molecular Biology, Green Publishing Associates and Wiley Interscience, NY (1989). Alternatively, the polynucleotides and vectors of the invention can be reconstituted to liposomes for delivery to target cells. The vectors containing the polynucleotides of the invention (for example, the variable heavy and / or light domain (s) of the immunoglobulin chains encoding sequences and the expression control sequences) can be transferred to 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, while calcium phosphate treatment or electroporation can be used for other cellular hosts; see Sambrook, supra. The invention further relates to a host transformed or transfected with the vector of the invention. Said host cell can be a prokaryotic or eukaryotic cell. The polynucleotide or vector of the invention that is present in the host cell can be integrated into the genome of the host cell or can be maintained extrachromosomally. The host cell can be any prokaryotic or eukaryotic cell, such as a bacterial, insect, fungal, plant, animal or human cell. Preferred fungal cells are, for example, those of the genus Saccharomyces, in particular those of the species S. cerevisiae. The term "prokaryotic" is intended to include all bacteria that can be transformed or transfected with a DNA or RNA molecule for the expression of an antibody of the invention, or the corresponding immunoglobulin chains. Prokaryotic hosts may include gram-negative bacteria as well as gram-positive bacteria such as, for example, E. coli, S. typhimurium, Serratia marcescens and Bacillus subtilis. The term "eukaryotic" is intended to include yeast cells, higher plants, insects and, preferably, mammals. Depending on the host employed in a procedure of recombinant production, the (poly) peptides / antibodies or the immunoglobulin chains encoded by the polynucleotide of the present invention can be glycosylated or can be non-glycosylated. The antibodies of the invention or the corresponding immunoglobulin chains can also include an initial amino acid residue of methionine. A polynucleotide of the invention can be used to transform or transfect the host, using any of the techniques commonly known to those of ordinary skill in the art. Additionally, methods for preparing fused genes, operably linked and expressing them, for example, in mammalian cells and bacteria, are well known in the art (Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989). The genetic constructions and the methods described here can be used to express the (poly) peptide / antibody of the invention or the corresponding immunoglobulin chains in eukaryotic or prokaryotic hosts. In general, expression vectors containing promoter sequences that facilitate efficient transcription of the inserted polynucleotide are used in relation to the host. The expression vector typically contains an origin of replication, a promoter and a terminator, as well as specific genes that are capable of providing phenotypic selection of the transformed cells. In addition, transgenic animals, preferably mammals, comprising cells of the invention can be used to »» * __. * A &. * .. ¡¿¿? ^: ._. .. ^^^^^^ ^ ^^^^^^^^^^^^^^^^^^^ 3 ^ ^^^^^^^^^^^^^ j ^ & the large-scale production of the (poly) peptide of the invention. Transformed hosts can be developed in fermenters and cultured according to techniques known in the art, to obtain optimal cell growth. Once expressed, the (poly) peptides / whole antibodies, their dimers, the individual light and heavy chains or other forms of immunoglobulin of the present invention can be purified according to procedures customary in the art, including precipitation with ammonium sulfate. , affinity columns, Column chromatography, gas electrophoresis and the like; see Scopes, Protein Purification, Springer-Verlag, N.Y. (1982). The antibody or its corresponding immunoglobulin chain (s) of the present invention can then be isolated from the growth medium, cell lysates or membrane fractions.

The isolation and purification, for example, of the antibodies or immunoglobulin chains, expressed microbially, of the invention, can be by any conventional means, such as, for example, separations by preparative chromatography and immunological separations, such as those involving the use of monoclonal or polyclonal antibodies directed, for example, against the constant region of the antibody of the invention. It will be apparent to those skilled in the art that the antibodies of the present invention can be coupled in addition to other portions, for example, specific target applications for drugs and imaging. This coupling can Siaa ^ SSSsSÉfefe ^ tí ^? Tí ^^^ Jíá ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ ^^^^^^^^^^^^^^^^^^^^^^^^^^^^ carried out chemically after the expression of the (poly) peptide / antibody or the antigen can be engineered to the binding site of the coupling product in the (poly) peptide / antibody of the invention, at the DNA level. Then the DNA is expressed in a suitable host system, and the expressed proteins are collected and renatured, if necessary. The invention further relates to a method for producing a (po I i) peptide encoded by the nucleic acid molecule of the invention, which comprises culturing the host of the invention, under suitable conditions, and isolating said (poly) peptide from the culture. The culture of said host cells, in general, is described further back, and can be carried out in accordance with established protocols. The same can be applied for the isolation of (poly) peptides. Additionally, the invention relates to a (poly) peptide that is encoded by a nucleic acid molecule of the invention, or can be produced by the method of the invention. The invention also relates to an antibody, or a fragment thereof or a derivative thereof, comprising at least one (poly) peptide of the invention. It is preferable that the antibody of the invention comprises the complete V H and V L chains, referred to above, together with the appropriate constant regions, such as the μ,?, A, K or α chain.

Specific applications of the antibody or fragment or derivative of the invention include the following: • Mature T lymphocytes can be functionally affected by structural or functional TCR blocking, as a result of antibodies or antibody derivatives specifically binding to the zeta chain, or by virtue of whether the biologically or pharmaceutically active molecules are specifically directed to the T cell surface by the anti-zeta chain antibodies or the antibody fragments / derivatives. Additionally, the development and selection of thymocytes can be affected by functional blocking of TCR or pre-TCR, as a result of antibodies or fragments / antibody derivatives specifically binding to the zeta chain. As the zeta chain is consistently expressed throughout the development of the T cell, from most immature thymocytes to mature T lymphocytes, the molecule can be useful to direct it to a wide variety of T-cell malignancies. NK cells they may also be functionally affected by structural or functional blockade of FcγRIIIA, as a result of antibodies or antibody derivatives specifically binding to the zeta chain, or by virtue of whether the biologically or pharmaceutically active molecules specifically target the NK cell surface by antibodies ? ^^^^^^ a ^ m ^^^^ _ ^^ __ ^ l ^^ __ ^ ____ ^^ _ ^^^^^^^^^^^^^^^^^^^^^ ^^^^^ k ^^^^^^^^^^^^^^^^^ ^^^^^^^^^^ chain anti-zeta or fragments of antibody or their derivatives. The antibody or its fragments or derivatives, among other things, can be a monoclonal antibody (semi-synthetic) or classically developed. The fragments include Fab 'or F (ab) 2 fragments. The antibodies of the present invention can comprise another domain, said domain being linked by covalent or non-covalent ligatures. Binding can be based on genetic fusion according to methods known in the art and described above, or can be effected, for example, by chemical binding as described, for example, in WO 94/04686. The additional domain present in the fusion protein comprising the antibody of the invention can be preferably linked by means of a flexible linker, advantageously a polypeptide linker, wherein the polypeptide linker comprises a plurality of hydrophilic amino acids, attached to peptide, with a length sufficient to cover the distance between the terminal end C of the additional domain, and the terminal end N of the antibody of the invention, or vice versa. The fusion protein described above may additionally comprise a cleavable linker or a cleavage site for the proteinases. These separating portions, in turn, may be insoluble or soluble (Diener and co-authors, Science, 231: 148, 1986) and may be selected to allow the release of the Drug from the ^^^^^ _ ^^^^^^ _____ ^^^^ _ ^ _ ^^^^^^^^^^^^^^^^^^ & ^^^^^ & ^^ - »q. < gi i? i antigen at the destination site. Examples of therapeutic agents that can be coupled to the antibodies of the invention, for immunotherapy, are: drugs, radioisotopes, lecithins and toxins. The drugs that can be conjugated to the antibodies of the present invention include compounds that are classically named drugs, such as mitomycin C, daunorubicin and vinblastine. When using radioisotopically conjugated antibodies of the invention, for example, for immunotherapy, certain isotopes may be more preferable than others, depending on factors such as the distribution of leukocytes, as well as stability and emission. Depending on the autoimmune response, some transmitters may be preferable to others. In general, radioisotopes emitting alpha particles and beta particles are preferred in immunotherapy. High-energy and short-range alpha emitters, such as 212Bi, are preferred. Examples of radioisotopes that can be linked to antibodies, antigens or epitopes of the invention, for therapeutic purposes, are: 125l, 131l, 90Y, 67Cu, 212Bi, 212At, 211Pb, 47Sc, 109Pd and 188Re. Other therapeutic agents that can be coupled to the antibody, antigen or epitope of the invention, as well as therapeutic protocols ex vivo and in vivo are known, or can be readily determined by those of ordinary skill in the art. Whenever appropriate, persons skilled in the art can use a polynucleotide of the invention that encodes any of the antibodies, antigens, or antigens.

¡^^ «¿^ ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ In a preferred embodiment, the antibody of the invention is a monoclonal antibody. In another preferred embodiment of the invention, the antibody is a bispecific antibody. In a preferred embodiment of the bispecific antibody of the invention, the first specificity is for the extracellular domain of the human zeta chain on the surface of an intact cell, and the second specificity is for an optionally different molecule, on the surface of a T lymphocyte. , a natural killer cell or a precursor of it. The bispecific antibody of the invention can bind to the destinations referred to above, which may be located in the same cell or in different cells. Said different cells can be, for example, two different T-lymphocytes of the same type, or a T lymphocyte and a natural killer cell or precursors of the foregoing, respectively. In another preferred embodiment of the bispecific antibody of the invention, the first specificity is for the extracellular domain of the human zeta chain on the surface of an intact cell, and the second specificity is for a different molecule on the surface of a different cell, from preference of a cell different from a T cell, an NK cell or a precursor thereof. It is preferred that this molecule be an antigen encoded in a virus, a '^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ ^^ _ ¡mjj ^^^^^^^^^^^^^ k ^^^ antigen associated with tumor, or a surface antigen, either in cells that present antigen (APC), very preferable, dendritic cells , or in cells that do not present antigen (non-APC) A preferred application of the bispecific antibody of the invention is to redirect the T lymphocytes against target cells, specifically directing, simultaneously, the zeta chain and a cell surface antigen. destination, with a bispecific antibody, instead of targeting the T lymphocytes on target cells, transfecting them with a chimeric zeta chain receptor (Romeo, Cell 64 (1991) 1037-1046). Since the bispecific antibody binds to the natural zeta chain, which is associated with the other TCR subunits, the signaling machinery of the entire TCR complex can be recruited in that way. In contrast, chimeric zeta chain receptors are not associated with endogenous TCR subunits and, thus, can only recruit the signaling effect isolated from the zeta chain, which appears to be insufficient to activate inactive T cells, or can cause alteration unbalanced functional status of T lymphocytes, with respect to the induction of activation against apoptosis. A preferred application of T cell-specific redirection comprises lysis of target cells, directing on them the cytotoxic activity of cytotoxic T lymphocytes. Another preferred application of T-cell specific redirection comprises sensitization of naive T lymphocytes, interlacing their zeta chain molecules with a surface antigen in the Éfá ^^^^^^ J ^^ - Iéjá & i ^^^^^^^^^^ - ^^^^^ - ^^^^^^ rh 32 antigen presenting cells (APC) or in cells no-APC, which have been modified to give sufficient co-stimulatory signals. On the other hand, naive T cells can be anergized or depleted by specific redirection, mediated by zeta chain, on 5 cells that do not provide sufficient costimulatory signals. Another preferred application of T-cell specific redirection comprises the induction of apoptosis in mature, activated T lymphocytes by re-coupling TCR mediated by strong zeta chain, thus mimicking the mechanisms that measured peripheral T cell tolerance, and contribute to peripheral immunological homeostasis. The thymic selection of thymocytes during the development of T cell can be modified by bispecific antibodies, by means of the entanglement of the zeta chain in the thymocytes, with surface molecules in the thymic cells that present antigen, directly involved in the process of positive and negative selection. Yet another preferred application of the bispecific antibody of the invention comprises activity-specific redirection Cytotoxic NK cells against target cells, specifically targeting, simultaneously, the zeta chain and a target cell surface antigen, with a bispecific antibody, instead of targeting the NK cells on target cells, by transfection of a chimeric zeta chain receptor (Tran, J. Immunol., 155 (1995) 1000-1009). ^^ _? ^^^^^^^^^ S ^ __ ^ ____ ^^^ g ^ ^^^^^^ ^^^^ _ ^^ _? ^ _ ^^^^^ _ ^ ___ ^^ l ^^^ ______ ^^^^^ _ ^^^^^^^^^ j ^^^ J ^^ In another preferred embodiment, the antibody derivative of the invention is a scFv chain In a preferred embodiment more of the monoclonal antibody of the invention, said antibody is an IgM. The invention further relates to a bispecific receptor comprising a (poly) peptide of the invention and natural receptor, natural ligands or a derivative thereof, which interact with a surface molecule in the same cell or in another cell; preferably those receptors or ligands are: CD4, CTLA-4, B7-1, B7-2, LFA-3, ICAM-1, -2, -3 or chemokines such as MIP-1a, MIP-1β, RANTES or SDF- 1. It is contemplated that the applications of the bispecific antibody of the invention are also applied to the bispecific receptor of the invention. The invention further relates to a pharmaceutical composition comprising the nucleic acid molecule of the invention, the vector, the host, the (poly) peptide, the antibody or the fragment or derivative thereof, and / or the bispecific receptor of the invention. The pharmaceutical composition of the present invention may additionally comprise a pharmaceutically acceptable carrier. Examples of acceptable pharmaceutical carriers are well known in the art and include phosphate, water, emulsion regulated solutions, such as oil / water emulsions, various types of wetting agents, sterile solutions, etc.

^^ The compositions comprising said carriers can be formulated by conventional known methods. These pharmaceutical compositions can be administered to the subject at a suitable dose. The administration of the appropriate compositions can be effected by different routes, for example, intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal. The dosage regimen will be determined by the attending physician and by clinical factors. As is well known in medical techniques, the dosages for any patient depend on many factors, including the size of the patient, the area of the body surface, age, the particular compound to be administered, sex, time and the route of administration, general health and other drugs that are being administered concurrently. A typical dose may be, for example, on a scale of 0.001 to 1000 μg (or of nucleic acid for expression or for inhibition of expression on that scale); however, doses below and above that exemplary scale are contemplated, especially taking into account the factors mentioned above. In general, the regimen, as a regular administration of the pharmaceutical composition, should be in the range of 1 μg to 10 mg per kilogram of body weight, per minute, respectively. The progress can be monitored by periodic determination. Doses will vary, but a preferred dose for intravenous administration of DNA is approximately 106 to 1012 copies of the DNA molecule. The compositions of the invention can be administered locally or systemically. The administration will generally be parenteral, for example, intravenous; DNA can also be directly administered to the target site, for example, by biolistic delivery to an internal or external target site, or by catheter, to a site in an artery. Preparations for parenteral administration include sterile, aqueous or non-aqueous solutions, suspensions and emulsions. Examples of non-aqueous solvents are: propylene glycol, polyethylene glycol, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspensions, including saline and regulated media. Parenteral vehicles include: sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present, such as, for example, antimicrobials, antioxidants, chelating agents and inert gases and the like. Additionally, the pharmaceutical composition of the invention may comprise other agents, such as interleukins or interferons, depending on the intended use of the pharmaceutical composition. It is contemplated by the present invention that the various polynucleotides and vectors of the invention are administered alone or in any combination, using vectors .,. ^. > __. f: i i &itai &ÉBi normal and / or gene delivery systems, and optionally, together with a pharmaceutically acceptable carrier or excipient. After administration, the polynucleotides or vectors can be stably integrated into the subject's genome. On the other hand, viral vectors can be used that are specific for certain cells or tissues, and persist in those cells. Suitable pharmaceutical carriers and excipients are well known in the art. Additionally, it is possible to use a pharmaceutical composition of the present invention comprising the polynucleotide or vector of the invention, in gene therapy. Suitable gene delivery systems may include: liposomes, delivery systems mediated by the recipient; Naked DNA and viral vectors, such as herpes viruses, retroviruses, adenoviruses and adeno-associated viruses, among others; see also supra. The delivery of the nucleic acids to a specific site in the body for gene therapy will also be achieved using a biolistic delivery system, such as that described by Williams (Proc. Nati, Acad. Sci. USA, 88 (1991) 2726-2729 ). The pharmaceutical compositions, methods and uses of the present invention can be conveniently used in humans, although the treatment of animals is also comprised by the methods and uses described herein. The invention relates to the use of the antibody of the invention in which the first specificity is for the domain extracellular of the human zeta chain, and the second specificity is for a different molecule on the surface of a T lymphocyte, a natural killer cell or a precursor thereof, for the preparation of a pharmaceutical composition for treating or preventing autoimmune diseases, immunological deficiencies , T cell malignancies, infectious diseases or for the suppression of the immune response, especially to avoid rejection of grafts after an organ transplant. In addition to the elimination of the target cells (e.g., tumor cells or virus-infected cells), the modes described in the present invention are coupling with cells (normal and malignant) of T-cell lineage, choosing the extracellular target as the target. zeta chain, can be used therapeutically to increase or suppress immunological responses and / or to influence T-cell disorders related to autoimmune diseases, immunodeficiencies or T-cell malignancies. Immunological suppression based on the extracellular targeting of the zeta chain ( and eta) can be used preferably to prevent graft rejection after transplantation. The modes described in the present invention for modifying signal transduction during T cell development and selection of the thymocyte by choosing the extracellular zeta chain as the extracellular destination can be used therapeutically to increase favorable immune responses, for example, in case of infectious diseases, tumors and immunodeficiencies, or for TO . JL m ........ ri ..., .. *. fc. < «WM ..,., £ -. < _. • :, Ai i? . to faithfully suppress unfavorable immunological responses, for example, in the case of autoimmune diseases or rejection of grafts after transplantation. Additionally the invention relates to the use of the antibody of the invention, where the first specificity is for the extracellular domain of the human zeta chain, and the second specificity is for a different molecule on the surface of a different cell, for the preparation of a pharmaceutical composition for treatment or prevention of malignancies, viral infections and other infectious diseases. The invention, additionally, relates to the use of the (poly) peptide or antibody or a fragment or derivative thereof, or the bispecific receptor of the invention, for the preparation of a pharmaceutical composition for increasing or suppressing NK cell-dependent immunity. or for the treatment of malignancies derived from NK cells. In addition to the elimination of target cells (for example, tumor cells or virus infected cells), the described ways of coupling NK cells by specific extracellular targeting of the zeta chain, can be used therapeutically to increase or suppress NK cell-dependent immunity, or to influence the malignancy derived from NK cell. Since the zeta chain is also expected to be expressed in the malignancies derived from the NK cell, the molecule may be additionally useful for directing it specifically to malignant NK cells, i? .. S A át? »ériím»? - -a-j? ¡Ma ^., - j ^ »¿I-fc < .J --..- * ,, »^ J? ._ AM _L ..., -. *. .-.,.. -AHA"-? As a destination. Additionally, the invention relates to a method for determining the expression of eta chain or zeta chain, in NK cells, T lymphocytes or their precursors, comprising '(a) contacting the (poly) peptide or the antibody or a fragment or derivative thereof, of the present invention, with NK cells, T lymphocytes or their precursors; and (b) determining the amount of bound (poly) peptide, antibody or derivative. The contact can be made by preferably incubating on ice the (poly) peptide or the antibody or a fragment or derivative thereof, with the NK cells, the T lymphocytes or their precursors, in a biological regulator, which resembles the conditions physiological (eg, phosphate buffered saline, PBS), for 20 to 40 5 minutes. After two washing steps with PBS, the (poly) peptide, the antibody or its fragments or derivatives, linked, for example, with an appropriate fluorescent labeled secondary antibody, can be detected and quantified by flow cytometric analysis , as described in example 4. The invention further relates to a kit or kit comprising the nucleic acid molecule, the vector, the host, the (poly) peptide, the antibody or a fragment or derivative thereof, and / or the bispecific receptor of the invention. Finally, the invention relates to a transgenic animal comprising, in its germ line, at least one tí ^^^^^^ _ ^^^^^^^^^^ _ ^ _ ^^^^^ ih ^^^^^^^^^^^? gi ^ t & ^^^^ tM ^^ ^^^^^^^^^^^^ _ ^^^? j ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ Transgenic animals can be produced according to conventional protocols such as those described, for example, in Palmiter, R. D., Brinster, R. L .: Germiine transformation of mice. Ann. Rev. Genet, 20 (1986) 465-499, and in Capecci M: Altering the genome by homologous recombination, Science 244 (1991) 1288-1292. Preferred examples of transgenic animals of the invention are: cattle, sheep, rabbits, mice 0 or rats. Table 1 shows series of sensitizers for PCR amplification of murine Ig heavy chain and light chain DNA fragments. The figures show: 5 FIGURE 1: Structure of TCR and early events in T cell activation. TCR consists of clonotypic chains (alpha + beta) and invariant chains (?, CD3 ?, CD3d and CD3e), with probable TCR of subunit composition a + ß, CD3 ed? e, ?? The location of the ITAM within the cytoplasmic domains of 0 the CD3 and? They are shown as small black ovals. A) In inactive T cells, the ITAMs are non-phosphorylated or only partially phosphorylated. The tyrosine-kinase protein Lck is associated with the cytoplasmic domain of CD4 or CD8. B) By interaction with an MHC-peptide complex, the TCR and CD4 or CD8 are coaggregated and the ITAM within the CD3 and? is it so phosphorylated by the Src-kinases Lck and / or Fyn. ZAP-70 or the related Syk kinase, binds specifically to ITAMs in which both tyrosine residues have been phosphorylated. After its recruitment to the TCR complex, ZAP-70 is activated by Lck, subsequently, after other molecules are thus recruited to the TCR complex and the activation proceeds to the additional downstream molecules (not shown), the P in a circle represents phosphorylated tyrosine residues. FIGURE 2: - ELISA analysis of murine Fab antibody fragments, selected by phage display, for binding to the extracellular part of the human zeta chain. The periplasmic preparations of soluble Fab fragments, expressed in E. coli, were incubated with immobilized zeta-peptide-BSA conjugate. Fab fragments specifically bound with an F (ab ') 2 fragment conjugated with horseradish peroxidase, of a goat anti-mouse IgG + IgM antibody were detected. The ELISA was developed by adding an ABTS substrate solution. Eight clones are presented per round of in vitro selection, on the x axis; OD values were measured by the ELISA reader at 405 nm and are presented on the y axis. For negative controls, the concavities were incubated with PBS instead of the periplasmic preparations. FIGURE 3.- Flow cytometric analysis of the specific binding activity to the zeta chain, of the antibody 2-B-5, on the surface of CD8 + T-lymphocytes and NK cells. 100,000 mononuclear peripheral blood cells were incubated from two healthy, different donors with undiluted cell culture supernatant of the hybridoma 2-B-5. Rat-specific antibody to the bound zeta chain was detected by goat anti-rat Ig antibody (IgG + IgM) conjugated to fluorescein (FITC), diluted 1: 100 in PBS. A triple color fluorescein analysis was carried out, applying a positive gate for CD8 + (tricolor) and a negative gateway for CD16 + (PE) cells, thus allowing the detection of fluorescence mediated with FITC (solid lines) attributed exclusively to CD8 + -T-lymphocytes (phenotype: CD8 +, CD16") without any contaminating signal of CD8 + - NK cells, similarly, triple color fluorescence analysis was carried out applying a positive gate for CD56 + - (PE) and a negative gate for CD3 + - cells (tricolor) thus allowing the detection of FITC-mediated fluorescence (solid lines) exclusively attributed to NK cells (phenotype: CD56 +, CD3 ') without any contaminating signal of CD56 + - T lymphocytes. As isotypic control (lines interrupted) the culture supernatant of an antibody with the same isotype (rat IgM) but with irrelevant specificity was used For the fixation of labeled cells 1% paraform was used aldehyde in PBS The cells were analyzed by flow cytometry in a FACS scanner (Becton Dickinson). FIGURE 4.- Results of a sandwich ELISA, confirming the reactivity of the antibody 2-B-5 with the natural zeta chain, present in the lysate of CD8 + T-cell lymphocytes. An antibody specific for the zeta chain, which recognizes amino acids 144-163 at the carboxy end of the human zeta chain, was applied for twelve hours to the concavities of a plate of 96 concavities, with U-bottom, followed by blocking for one hour at room temperature with PBS / 3% of BSA. The CD8 + lysate - undiluted cells were then added at various dilutions and incubated for one hour at room temperature. As a negative control, the concavities were incubated with PBS instead of the cell lysate. In the next step, purified antibody 2- B-5 was added at a concentration of 1 μg / ml and incubated for one hour. The bound 2-B-5 antibody was detected with biotinylated mouse anti-mouse IgM antibody, followed by an Avidin-peroxidase conjugate. The ELISA was finally revealed by adding an ABTS-substrate solution. The colored precipitate was measured at 405 nm, using an ELISA reader. FIGURE 5.- ELISA-based analysis for the specific binding to the zeta-peptide-KLH conjugate of the recombinant Fab fragment of the rat monoclonal antibody 2-B-5, expressed in the periplasm of E. coli. The coating of the zeta-peptide-KLH conjugate was carried out at 4 ° C for 12 hours, followed by a single washing step with PBS / 0.05% Tween. The concavities were blocked for one hour with PBS / 3% bovine serum albumin (BSA) and rewashed once. Then, periplasm preparations containing Fab, undiluted, and in various dilutions, were added and incubated for two hours. As controls Negative concavities were incubated with PBS instead of the periplasmic preparations. For detection of Fab fragments attached to the zeta-peptide-KLH conjugate, an antibody with murine anti-His tag was used, followed by a goat anti-mouse IgG antibody, polyclonal, conjugated with peroxidase. The ELISA was finally revealed by adding ABTS-substrate solution. The change of the colored substrate was measured by means of an ELISA reader, at OD 405 nm. FIGURE 6: DNA and protein sequence of the VH region of anti-zeta 2-B-5 chain antibody. The numbers indicate the nucleotide positions (nt), the amino acids (aa9 are presented in the single-letter code) The cells indicate the three CDRs FIGURE 1.- DNA and protein sequence of the VK region of the 2-B antibody -5 anti-zeta chain The numbers indicate the positions of the nucleotide (nt), the amino acids (aa) are presented in single-letter code The boxes indicate the three CDRs FIGURE 8.- Results of an analysis of incorporation of BrdU, based on ELISA, which detects the proliferation of cells carried out in order to determine the stimulation of CD8 + T cells, NK cells and PBMC induced by the anti-zeta chain 2-B-5 antibody. 96-well flat-bottomed microtiter with 2-B-5 antibody purified in several dilutions overnight at 4 ° C. Added in triplicate to concavities •• "'-' of a microtiter plate, 100,000 CD8 + - T lymphocytes, NK cells and non-separated PBMC, respectively To control the specificity of 2-B-5 mediated stimulation, an antibody of the same isotype was used (Rat IgM) with irrelevant specificity at the same concentrations The IKT3 antibody (isotype IgG2a) which recognizes the human CD3 complex was applied as a specific positive control for stimulation of T cells and PBMC without separating, respectively. a murine IgG2a antibody, of irrelevant specificity, as an isotope control for OKT3. A blank control (concavities without cells) and a background control (concavities with BrdU) were also included. After a three-day incubation period, the BrdU marker solution was added for 24 hours. After this the cells were lysed and fixed, after which a anti-BrdU antibody, which binds BrdU incorporated in the newly synthesized cellular DNA. This antibody conjugated with peroxidase was detected by the subsequent reaction of the substrate. The reaction product was quantified by means of an ELISA reader at a wavelength of 450 nm. FIGURE 9.- Cytometric analysis of the internalization flux of the TCR / CD3 complex, induced by the binding of the anti-zeta chain antibody. 200,000 mononuclear cells were incubated with the anti-zeta 2-B-5 antibody, at a concentration of 1 μg / ml, at 4CC or 37 ° C for 30 or 60 minutes. HE carried out a parallel experiment with an anti-CD3 antibody rat human. The coronation process was completed by washing twice with cold PBS. To label the antibody bound to the cell surface, the cells were incubated with fluorescein-conjugated goat anti-rats Ig (IgG-IgM) antibody (FITC), diluted 1: 100 in PBS. Only the secondary antibody was used as a negative control. FIGURE 10.- DNA and protein sequence of the single-chain, bispecific, anti-zeta / anti-EpCAM antibody. The numbers indicate the nucleotide positions (nt), the resulting amino acid sequence is illustrated below the nucleotide sequence. The DNA sequence encoding the antibody is started at position 67 and ends at position 1605. Nucleotides 10 to 66 encode a leader peptide, which mediates the secretion of the bispecific antibody in mammalian cells. The first six nt (position 1 to 6) and the last six nt (position 1632 to 1637) contain the recognition sites of the restriction enzyme for EcoRI and Sali, respectively. FIGURE 11.- Cytotoxic activity of PBMC and CD8 + T-lymphocytes, redirected against Epito-positive Kato cells by the bispecific anti-zeta / anti-EpCAM antibody. 200,000 PBMC or CD8 + T-lymphocytes, unstimulated, in a volume of 100 μl, were added to 10,000 Kato III cells labeled with chromium 51 in a volume of 100 μl. The bispecific antibody was added in concentrations of 40 ng / ml to 5 μg / ml in a volume of 50 μl. The microtiter plates were incubated for 16 hours at 37 ° C, 5% CO2. After the incubation period, 50 μl of fc »l * A é, i - .. -. .. .. * .. * ...- «-. I- i. ^^ .- A ^^ - J-.A ^,. ^ J _ ^ - ^ ^. ^^ ._.____ ^ _ ^ __ ^ _sá ____-- = í __________ supernatant of each concavity, and analyzed for the 51Cr released, in a gamma counter FIGURE 12.- Cytotoxic activity of NK cells, redirected against EpCAM-positive target cells, by the anti-bispecific antibody 5 zeta / anti-EpCAM chain. 100,000 NK cells in a volume of 100 μl were added to 10,000 Kato III cells labeled with chromium 51 in a volume of 100 μl. The bispecific antibody was added at a concentration of 1 μg / ml in a volume of 50 μl. The microtiter plates were incubated for 4 hours. hours at 37 ° C, 5% CO2. After the incubation period 50 μl of supernatant was removed from each concavity and analyzed for the 51Cr released, in a gamma counter. These and other modalities are described and are comprised by the description and examples of the present invention. Other literature that relates to any of the antibodies, methods, uses and compounds to be employed in accordance with the present invention can be obtained from public libraries and databases, using, for example, electronic devices. You can use the public database "Medline", which is available on the Internet, for example, at http://www.ncbi.nlm.nih.gov/PubMed/Medline.html. Other databases and addresses, such as http://www.ncbi.nim.nih.gov/, http: // www. infobiogen.fr/, http://www.fmi.ch/biology/research__tools.html, http: // www.tigr.org/ are known by the experts in the field and can also be obtained using, for example, http://www.lycos.com A summary of patent information in biotechnology and a view of relevant sources of patent information useful for retrospective research and to be currently aware, is given in Berks, TIBTECH 12 (1994) 352-364. The foregoing description generally describes the present invention. More complete understanding can be obtained by reference to the following specific examples which are given herein for illustrative purposes only, and are not intended to limit the scope of the invention. The examples illustrate the invention.

EXAMPLE 1 IMMUNIZATION OF MICE WITH THE ZETA-PEPTIDE-KLH CONJUGATE AND DETERMINATION OF THE SERUM TITLE USING THE ZETA-PEPTIDE-BSA CONJUGATE F1 crossbull c Balb / c x C57 black mice, immunized at 10 weeks of age, were immunized with the zeta-peptide-KLH conjugate (jerini Bio Tools, Berlin). The peptide was coupled with the amino acid sequence (QSFGLLDPKLC) of the N-terminus of the zeta chain, with KLH activated with maleinimide, directly, by the mercapto group of the C-terminal cysteine. The conjugate was dissolved in 0.9% NaCl at a concentration of 100 μg / ml. The 1: 2 solution was subsequently emulsified with complete Freund's adjuvants and 50 μl per mouse was injected intraperitoneally. The mice received __ «____ __ ^ ___________ i __ * < ____ booster immunizations after 4, 8, and 12 weeks, in the same manner, except that complete Freund's adjuvants were replaced by incomplete adjuvants. Ten days after the first booster immunization, blood samples were taken and the serum titer of the antibody against the zeta-peptide-BSA conjugate was tested by ELISA. The serum titer was more than 100 times higher in immunized animals than in non-immunized animals. Three days after the second boost, spleen cells were fused with P3X63Ag8.653 cells (ATCC CRL-1580) to generate hybridoma cell lines, following common and current protocols, such as those described in Current Protocols in Immunology (Coligan, Kruisbeek, Margulies, Shevach and Strober, Wiley-lnterscience, 1992). After fusion with PEG, cells were seeded at 100,000 cells per concavity in microtiter plates and developed in 200 μl of RPMI 1640 medium, supplemented with 10% fetal bovine serum., 300 units / ml of human interleukin 6, recombinant, and HAT additive for selection. The culture supernatants of densely developed concavities were tested by ELISA analysis at 1:20 dilution. The ability of the hybridoma supernatants to bind to the zeta-peptide-BSA conjugate was tested by the following ELISA: 100 μl of conjugated zeta-peptide was applied to bovine serum albumin, in the same manner as described for the zeta-conjugate. Peptide-KLH (Jerini Bio Tools, Berlin), to the walls of a plate of 96 concavities with U-bottom (Nunc, maxisorb) a .? i ... ». £, -H-_ .---. al - »« * _ «J a concentration of 5 μg / ml. The coating was carried out overnight at 4 ° C, after washing three times with washing buffer (0.1M NaCl, 0.05M Na2HPO4, pH 7.3, 0.05% Tween 20, 0.05% NaN3) the following was made blocking with 200 μl of 2% skimmed milk powder, added to the washing regulator for one hour at room temperature. In the next step, supernatant of pure hybridoma was incubated and at various dilutions, for two hours, at room temperature. As a detection system, polyclonal antibody conjugated with diluted horseradish peroxidase was used against mouse immunoglobulin. After washing five times the ELISA was finally revealed by adding the TMB-substrate solution (tetramethylbenzylphine, Boehringer Mannheim). The colored precipitate was measured after 15 minutes at 405 nm using an ELISA reader. Supernatants of 150 clones that exhibit strong ELISA signals were selected for flow cytometric analysis. To check the binding activity of the hybridoma supernatants on T lymphocytes and NK cells, a flow cytometric analysis was carried out. 1 x 106 PBMC were incubated for 30 minutes on ice in 50 ul of undiluted supernatant from 150 different clones, respectively and bound antibodies were detected subsequently by a F (ab ') 2 conjugated to fluorescein (FITC) , FROM AN ANTIBODY rabbit anti-mouse Ig (Dako Hamburg, Code No. F0313), diluted 1: 100 in PBS. To avoid non-specific binding in the following steps ^^^^^^^^ ^^ j & ^^^ labeling or marking, the free valences of the FITC-conjugated antibody were blocked by addition of 50 .mu.l of mouse serum diluted 1:10 (Sigma Immunochemicals, Deisenhofen , M-5905) for 30 minutes. To distinguish the two PBMC 5 subseries, the previously labeled cells were divided. Half was coated with a conjugated anti-CD8 antibody, tricolor, diluted 1: 100 (Caltac Laboratories, Buriingame, E.U.A., Clave No. MHCD0306); the other half was stained with an anti-CD56 antibody conjugated with phycoerythrin (PE) diluted 1:25 (Becton Dickinson, Heidelberg, Catalog No. 347747). As a negative control, monoclonal murine antibody of irrelevant specificity was used instead of the hybridoma supernatants. Anti-CD16 and anti-CD6 unlabeled antibodies were used for specific staining of NK cell and T lymphocytes, respectively, to control the passage of primary labeling. 5 The cells were analyzed by flow cytometry in a FACS scanner (Becton Dickinson, Heidelberg). was performed with FACS stained and fluorescence intensity was measured, as described in Current Protocols in Immunology (Coligan, Kruisbeek, Margulies, Shevach and Strober, Wiley-Interscience, 1992). 0 was performed fluorescence analysis bicolor applying a positive gate for CD8 + and CD56 +, respectively, thereby allowing the detection of FITC-mediated fluorescence separately on CD8 + T and NK -linfocitos cells. Despite a clear staining of CD8 + -K-lymphocytes and 5 NK cells by the respective positive control antibodies, & jg & ei 'g ^^^^^^^^ & ^ & ^^ Í none of the 150 hybridoma supernatants showed binding activity on CD8 + T -linfoc cough and / or NK cells?.

EXAMPLE 2 IN VITRO SELECTION for anti-zeta bank ANTIBODIES Murine combinational by the method phage display A 10-week-old Balb / cx C57 black crossover mouse was immunized as described in Example 1. Ten days after the first booster immunization, blood samples were taken and the antibody anti-serum titer was tested. the zeta-peptide-BSA conjugate, by ELISA (see example). The serum titer was greater than 1000 times higher in the immunized animals, compared to unimmunized animals. Three days after the third injection the murine spleen cells were harvested. To isolate the total RNA, a protocol according to Chomczynski (Analytical Biochemistry 162 (1987) 156-159) was used. A DNA library encoding murine immunoglobulin (Ig) kappa light chains and Ig heavy chain Fd fragments (= VH + CH1) was constructed by RT-PCR in murine spleen RNA, respectively. CDNA was synthesized according to normal protocols (Sambrook, Cold Spring Harbor Laboratory Press 1989, second edition). Sensitizing series were selected (illustrated in: i * • & Á. & Mtíí? R'h fe -Man-- - •. -. . jaajaat.j '. t. ^ t? i u? titi ^?. ^ _ AÁ ^ .. ^^ me, í¡i ^ Á? T ^ s ^, _ .. & Figure 1), resulting in a 5'-Xhoi recognition site and a 3'-Spel recognition site for the heavy chain, and a 5'-Sacl recognition site and a 3'-Xbal recognition site for light chain fragments. For the PCR amplification of the 5 HC DNA fragments, each of eight different specific sensitizers for the 5'-VH family was combined with four 3 'sensitizers, which hybridize to the 3' region of the HC-CH1 domain of different subclasses of IgG; for the PCR amplification of the kappa light chain fragments each was combined of seven different sensitizers specific for the 5'-VK family, combined with a 3 'sensitizer, hybridizing to the 3' end of the kappa constant region (CK). The following PCR program was used for the amplification: initial denaturation at 94 ° C for 2 minutes; 40 cycles of amplification: denaturation at 94 ° C for 20 seconds; first fixation at 52 ° C for 50 seconds, and first extension at 72 ° C for 60 seconds, followed by a final extension of 10 minutes at 72 ° C. 450 ng of the kappa light chain fragments were ligated (digested with Sacl-Xbal, large fragment) (Barbas and co-authors, Proc. Nati, Acad. Sci. USA 88, 7978-82 (1991)). The resulting light chain bank was then transformed into 300 μl XL1 blue of electrocompetent Escherichia coli, by electroporation (2.5 kV, 0.2 cm separation cell, 25 FD, 200 ohm, pulsator of Biorad genes), which resulted in a bank size of 6 x 108 independent clones. After one hour of phenotype expression, positive transformants were selected for their resistance to vector-encoded carbenicillin in 100 ml of liquid SB subculture overnight. The cells were then harvested by centrifugation and the plasmid preparation was carried out using equipment commercially available for the preparation of plasmid (Qiagen). 2800 ng of this plasmid DNA containing the kappa light chain bank (digested with Xhol-Spel, large fragment) was ligated with 900 ng of the HC-Fd-DNA fragments (digested with Xhol-Pel) and transformed again to two 300 μl aliquots of XL1 blue of electrocompetent E. coli, by electroporation (2.5 kV, 0.2 cm separation cell, 25 FD, 200 ohm), which resulted in a combinatorial library of antibody Fab fragments, which consisted of 4 x 108 independent clones. After one hour of phenotype expression, the positive transformation was selected by resistance to carbenicillin. After this adaptation, these clones were infected with an infectious dose of 1 x 1012 particles of the helper phage 20 VCSM13, which resulted in the production and secretion of the filamentous M13 phage, each phage particle containing a copy of a single strand of the vector phagemid encoding a single murine antibody Fab fragment, and exhibiting the corresponding Fab protein on the surface of the phage. The 25 Fab fragments were anchored on the surface of the fab by a translational fusion ^^^^^^^^^^? ^^^ ^ ^^^^^ _ ^ _ ^ _ ^^ ____ ^^^ __________________ ^^ __ ^^^^ ^^^^^^^^^^^ ^^^^^ ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ of the phage, spontaneously associating the Ig light chain with the HC fragment. This phage bank bearing the cloned Fab 5 repertoire was harvested from the culture supernatant by precipitation of PEG8000 / NaCl and centrifugation, redissolved in RPMI 1640, medium supplemented with 10% FCS and incubated with zetapeptide-BSA conjugate. immobilized or with CD8 + - isolated lymphocytes or NK cells in an order that alternates daily. The two were isolated subpopulations of human PBMC with anticipation by an immunomagnetic separation method. Mononuclear cells obtained from peripheral blood by Ficoll density gradient centrifugation were first incubated with a specific primary antibody of murine origin, directed against CD8 or CD56 human and was subsequently subjected to rosetting with paramagnetic beads (Dynal, Oslo, Norway), conjugated with a sheep anti-mouse IgG antibody. The CD8 + cells - T cells and CD56 + - NK cells were isolated with a magnet fixed to the wall of the tube containing the cell suspension. After incubating the bank of phages with CD8 + - purified T lymphocytes or with NK cells for two hours at 4 ° C under continuous stirring, respectively, rescued the phage-bound particles of unbound phage particles from the paramagnetic beads by means of the paramagnetic beads. fixed to the cells. Consequently, the exhibition was also carried out to the magnetic field in order to get rid of the washing solution that ^^ ^^^^^^^^^^^^^^^^^^^ i ^^^^^^^^^^^^^^^^^^^^^^^^ ^^^^^^^^^^^^^ ^ ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ suspend the cells and thus reduce the phage background. Finally, the specifically bound fab particles were eluted from the cells by HCl-glycine, pH 2.2 and after neutralizing with 2M Tris pH 12, the eluate was used for infection of a new XL1 blue culture of new E. coli, without infection. The transduced cells were successfully selected again with a copy of pGomb3H phagemid, which encodes a murine Fab fragment, for its resistance to carbenicillin and subsequently infected with the auxiliary phage VCMS13 to start another round of antibody and selection display. in vitro The complete in vitro selection procedure consisted of two initial rounds of scanning on immobilized zeta-peptide-BSA conjugate, followed by a round of scanning on CD8 + - T lymphocytes and, subsequently, on CD56 + - NK cells, respectively. Then two more rounds of scanning were carried out on immobilized conjugate zeta-peptide-BSA, followed again by a round of scanning on CD8 + - T cells and, finally, on CD56 + - NK cells, respectively. Scanning on immobilized antigen was carried out as described (Barbas and coauthors, Proc. Nati, Acad. Sci. USA 88, 7978-82 (1991)), in order to maintain the selection pressure on the Fab fragments specific for the zeta chain. , which may otherwise be lost by non-specific elution of phage particles from cell surfaces containing a large number of different antigens, in addition to the zeta chain After each round of scanning, the plasmid DNA was prepared from the resulting culture of E. coli. For the production of soluble Fab proteins, the gene III DNA fragment is excised from these plasmid-DNA preparations (Spel / Nhel), thus destroying the translational fusion of the Fab segment with the gene III protein. After religation, this plasmid DNA assembly was transformed to 100 μl of competent E. coli XL1 blue, heat shock, and plated on Carbenicillin LB-agar. Individual colonies developed in 10 ml cultures of Lb-Carb / 20 mM MgCl 2 and Fab expression was induced after six hours, adding isopropyl-β-D-thiogalacoside (IPTG) to a final concentration of 1 mM. These cells were harvested after 20 hours, centrifuged and through four rounds of freezing at -70 ° C and defrosting at 37 ° C, the outer membrane of the bacteria was destroyed by temperature shock so that soluble periplasmic proteins, including the Fab antibody fragments, are released into the liquid. After removing the intact cells and cell debris, the supernatant was tested by ELISA for the Fab antibody fragments that bind to the zeta-peptide-BSA conjugate. The detection of the Fab fragments bound to the immobilized zeta-peptide-BASA conjugate was carried out using a F (ab ') 2 fragment conjugated with horseradish peroxidase, from a IgG antibody + goat anti-mouse IgM (0.16 μg / ml) (Pérèse, Rockford, E U. A., Product No. 311448) The signal was developed by adding a substrate solution containing acid 2., 2'-azino-bis (3-ethylbenz-thiazolin-6-sulfonic acid) and sodium perborate, and was detected at a wavelength of 405 nm. In contrast to the clones taken from the bank prior to in vitro selection, those clones tested after different rounds of scanning proved to be positive in the zeta-peptide ELISA with an increasing overall frequency until the seventh sweep round, which was performed with CD8 + - purified T cells (figure 2). However, from the seventh to the eighth rounds of scanning, the last of which was carried out with purified NK cells, the number of positive clones dropped significantly. This indicates that the clones that could be enriched by virtue of their binding activity on T cells, were mostly not correct with NK cells with the only exception of clone 90 still present after the final sweep step on NK cells. To verify the binding activity of the Fab fragments reactive to zeta-peptide, a flow cytometric analysis was carried out on CD8 + T-cells and NK cells. 1 x 106 PBMC were incubated in 50 μl of undiluted periplasmic preparation, for 30 minutes, on ice, followed by incubation with a fluorescein-conjugated F (ab ') 2 fragment (FITC) of an anti-mouse IgG + IgM antibody. goat (Jackson Immunoresearch Laboratories, West Grove, USA, Key No. 115-096-068) diluted 1: 100 in PBS. To avoid non-specific binding during the following steps of > TO? A i? A? . _. • -i + * > .. ____-_._____________________________ ^ __________________ ^ _ ^^ labeling, the free valences of the FITC antibody were blocked by adding 50 μl of mouse serum diluted 1:10 (Sigma Immunochemicals, Deisenhofen, M-5905) for 30 minutes. To distinguish the two PBMC subseries, the previously labeled cells were divided. One half was stained with a conjugated anti-CD8 antibody, tricolor, diluted 1: 100 (Caltac Laboratories, E.U.A., Key No. MHCD0306); the other half was stained with an anti-CD56 antibody conjugated with phycoerythrin diluted 1:25 (Becton Dickinson, Heidelberg, catalog No. 347747). As a negative control, included periplasmic preparations of Fab antibody, of particular specificity. Anti-CD16 and anti-CD6 unlabeled antibodies were used as positive controls for NK and CD8 + cells - T lymphocytes, respectively. Two-color fluorescence analysis was carried out in a FACS explorer (Becton Dickinson) applying a positive gate for CD8 + and CD56 + cells, respectively, thus allowing the detection of fluorescence mediated by FITC, separately in CD8 + T-lymphocytes and NK cells. Cell labeling and fluorescence measurements were performed as described in Current Protocols in Immunology (Coligan, Kruisbeek, Margulies, Shevach and Strober, Wiley-lnterscience 1992). Using the protocol mentioned above, 60 different clones were analyzed in the PBMC, which showed that they reacted with the zeta-peptide-BSA conjugate in the ELISA analysis. Despite the positive, clear FACS signals, displayed by positive controls, ___? J? _g »___ g __ ^ g ____ ^ __ none of the periplasmic preparations of the anti-zeta chain Fab antibody fragments could demonstrate that it binds on the surface of CD8 + T-lymphocytes of NK cells. This result can be explained by the low affinity interactions of the Fab fragments selected by the scanning procedure with the surface of the T cells, which may be sufficient for enrichment during in vitro selection, but below the sensitivity of the cytometric analysis flow. However, the disappearance of zeta-peptide-reactive clones during the final selection step, carried out on NK cells, strongly indicates that these clones potentially reactive with T cells do not bind at all to the surface of NK cells. On the other hand, clone 90, which appeared first during the final round of scanning on NK cells, was determined by comparing its variable region sequences with those of other zeta-peptide-rectified clones that appeared previously during in vitro selection, very probably exhibited low affinity binding to the surface of the NK cell without interacting with the T cells. Consequently, clone 90 did not appear before the second round of scanning on NK cells and no binding signal was detectable on the T cells nor was it detectable. in NK cells by cytometric flow analysis of the corresponding Fab antibody fragments.

EXAMPLE 3 IMMUNIZATION OF RATS WITH THE CONJUGO ZETA-PÉPTI DO-KLH, gj ^^^ wiiy? s ^^^^? i ^^^^^ t ^^^^^^? gfcgNtíi ^^^ i AND PRODUCTION OF ANTI-ZETA ANTIBODIES THROUGH HYBRIDIZATION TECHNOLOGY At the age of three months, a Sprague Dawley rat was immunized with Jurkat of human T cell line (ATCC TIB-152) by intraperitoneal injection of 1 x 10 7 cells. Three months later the animal was immunized with the zeta-peptide-KLH conjugate. The conjugate was dissolved in 0.9% NaCl at a concentration of 200 μg / ml. The 1: 2 solution was emulsified with complete Freund's adjuvants and 100 μl was injected intraperitoneally and subcutaneously. The rat received a booster immunization after four weeks, in the same manner, except that no adjuvants were added. Three days after the booster the animal was sacrificed and the spleen cells were fused with P3X63Ag8.653 cells (ATCC CRL-1580) to generate hybridoma cell lines, following normal protocols. After the PEG fusion, the cells were seeded at 100,000 cells per concavity, in microtitre plates and developed in 200 μl of RPMI 1640 medium supplemented with 10% fetal bovine serum, and HAT additive for selection. After 8 days of culture the supernatant was completely removed and replaced with fresh medium. After another four days, the culture supernatant of each 1: 1 concavity was diluted and tested by ELISA (see example 1). The supernatants of those 45 concavities that exhibited the strongest reactions with the immobilized zeta-peptide-BSA conjugate were selected for analysis of FACS in CD8 + T lymphocytes and in NK cells, which was carried out as described in example 1 for murine monoclonal antibodies, except that an anti-rat immunoglobulin antibody labeled with FITC (IgG + IgM) was used (Dianova / Jackson, Hamburg, Catalog No. 112-016-044), instead of the F fragment ( ab ') 2 conjugated to FITC of a rabbit anti-mouse Ig antibody. Of the 45 different rat monoclonal antibodies tested, only one clone, designated 2-B-5, tested to bind to both CD8 + T-lymphocytes and NK cells. Therefore, this clone was analyzed in more detail, as described in examples 4 to 7.

EXAMPLE 4 CYTOMETRIC ANALYSIS OF FLOW OF ANTIBODY 2-B-5 ANTI-CHAIN ZETA, IN CD8 + -CELLS T AND IN NK CELLS In order to test the binding activity of the antibody 2-b-5 on the surface of CD8 + T-lymphocytes and NK cells, mononuclear cells were isolated from the peripheral blood of two different healthy donors, by Ficoll density gradient centrifugation. In each concavity of a titration microplate, 100,000 mononuclear cells were incubated, with the supernatant of the culture of undiluted cells, of the hybridoma 2-B-5, and with various dilutions thereof, respectively. As a negative control, the culture supernatant of an antibody with the same isotype (rat IgM) but of irrelevant specificity was used. After 30 minutes of incubation in ice cells, it was washed twice with PBS and subsequently stained with two different antibody labeling mixtures. CD8 + - T cells were simultaneously incubated for half an hour on ice with a fluorescein-conjugated goat anti-rat Ig (IgG + IgM) antibody (FITC) (Danova / Jackson, Hamburg, Catalog No. 112-016-044) , diluted 1: 100 in PBS a CD56 antibody conjugated with phycoerythrin (PE) (Becton Dickinson, Heidelberg, catalog No. 347747) diluted 1:25 in PBS and CD3 tricolor conjugated antibody (Caltac Laboratories, Buriingame, USA, No of key MHCD0306) diluted 1:50 in PBS. Marker serum (Sigma Aldrich, St. Louis, USA, Catalog No. 054H-8958) was added to this marker mixture, at a 1:10 dilution, to avoid non-specific binding reactions, anti-rat antibody to the mouse antibodies. 100 in PBS, an antibody conjugate tricolor CD8 (Caltac Laboratories, No. key MHCD0806) diluted 1: 100 in PBS and an antibody CD16 NK cell fraction with the same anti-rat Ig antibody goat incubated diluted 1 conjugated with phycoerythrin (PE) (Becton Dickinson, Heidelberg, Catalog No. 347617), diluted 1:25 in PBS. This mixture was supplemented with mouse serum as well. The labeled cells were washed twice in PBS before fixing with PBS / 1% paraformaldehyde. The cells were analyzed by flow cytometry in the FACS scanner (Becton Díckinson, Heidelberg). The ^^ ¿¿¿¿? ^ ^ M ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ ^ ji ^ l ^^^^^ stained and FACS measuring fluorescence intensity as described in current Protocols in Immunology (Coligan, Kruisbeek, Margulies, Shevach and Strober, Wiley-lnlerscience, 1992). Held fluorescence analysis of triple 5 color, applying a positive gate for CD8 + cells (Tricolor) and a negative gate for CD16 + (PE) cells, which allowed the detection of fluorescence FITC-mediated exclusively attributed to CD8 + . -linfocitos T (phenotype: CD8 +, CD16 ') without any contaminating CD8 + -cells signal NK Similarly was 10 out fluorescence analysis triple colored by applying a positive gate for CD56 + - (PE) and a gate negative for CD3 + cells (tricolor), allowing the detection of fluorescence FITC-mediated exclusively attributed to NK cells (phenotype CD56 +, CD3") without any contaminating signal CD56 + - 15 lymphocytes T. As shown in figure 3, the hybridoma antibody 2-B-5 binds specifically to the surface of T lymphocytes and that of NK cells, from different donors.

EXAMPLE 5 CONFIRMATION OF SPECIFICITY FOR THE ZETA CHAIN OF THE ANTIBODY 2-B-5, THROUGH ELBOW ELISA A sandwich ELISA was carried out in order to confirm the specificity for the zeta chain of the monoclonal antibody 2-B- - ^^^^^^^ tíá ^^ s ^^^^^^^^^^^^^^^^^^ mmmu ^^^^^^^^^ ^^^^^^ »» ^^^^^^^ ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ of CD8 + - purified T lymphocytes, which are known to express the zeta chain, and incubated with an immobilized antibody, which recognizes the intracellular zeta chain domain. It was then possible to capture zeta chain molecules of the cell lysate, by this antibody, and subsequently detected by the antibody 2-B-5 formed against the short extracellular portion of the zeta chain. The isolation of CD8 + -linocytes was carried out with paramagnetic beads, as described in example 1. The detailed steps were carried out according to the instructions of the manufacturers (Dynal, Oslo, Norway). It was subjected to CD8 + lysis - purified cells, by means of the detergent NP-40 (Sigma, Deisenhofen) in the presence of the protease inhibitor phenylmetanesulfonyl fluoride (PMSF) (Merck, Darmstadt). For detailed regulator formulation see Sambrook, Molecular Cloning: A Laboratory Manual, 2a. edition, Cold Spring Harbor Laboratory Press, Cold Springer Hobart, NY (1989). The sandwich ELISA was carried out as follows way: An antibody specific for the zeta chain was applied (Santa Cruz Biotechnology, Catalog No. 1124), which recognizes amino acids 144-163 at the carboxy terminus of the zeta chain, at concavities of a plate of 96 background concavities. U (Nunc, Maxisorb), at a concentration of 5 μg / ml. The coating overnight at 4 ° C, the following block was made with 3% BSA in PBS, for one hour, at room temperature. Subsequently the lysate of undiluted CD8 + cells was added and several dilutions were incubated for one hour. As a negative control, the concavities were incubated with PBS instead of the cell lysate. In the next step, purified monoclonal antibody 2-B-5 was added at a concentration of 1 μg / ml and incubated for one hour. The bound 2-B-5 antibody was detected with a biotinylated mouse anti-mouse IgM antibody (Zymed, San Francisco, CA, E.U.A., catalog No. 03-9840); working concentration 400 ng / ml), followed by an Avidin-peroxidase conjugate (Dako, Hamburg, code No. P 03347, working concentration 1 μg / ml). Finally the ELISA was revealed by adding a solution of ABTS-substrate (Boehringer Mannheim, Mannheim, Germany, Catalog No. 1682008). The colored precipitate was measured at 405 nm, using an ELISA reader. The rat IgM antibody 2-B-5 was purified from the hybridoma culture supernatant, by ion exchange chromatography, using a Bakerbond Abx column (JT Baker, Greisheim, Germany), according to the manufacturer's manual . As shown in Figure 4, the monoclonal antibody 2-B-5 binds to the zeta chain molecules from the T cell lysate captured by an immobilized antibody, which recognizes the intracellular zeta chain domain, with the signal from ELISA specifies for zeta, which strictly depends on the dilution of the . . __ ^^^^ ___ - J £, ¡^^^^^^^^^ and that varies distinctly above that of the negative control.

EXAMPLE 6 CLONING OF THE VARIABLE REGIONS OF ZETA ANTIBODY 2- B-5 AND EXPRESSION OF THE CORRESPONDING FRAGMENTO Fab IN E. coli RNA of 5 x 106 cells was isolated from the rat hybridoma cell line 2- B-5, according to the method described by Chomczynski and coauthors (Anal. Biochem., Volume 162, pages 156-9, 1989). The total RNA was reverse transcribed with the MMLV reverse transcriptase superscript II (Gibco BRL, Eggenstein) according to normal protocols (Sambrook, Cold Spring Harbor Laboratory Press, 1989, 2nd edition). Specific sensitization of the cDNA with the two oligonucleotides designated ratcmuRT was carried out (GTGCAGGGCCAGAGAAGGCATC), which coincides with a short sequence of the constant region of the rat mu and ratckRT chain (GTAGGTCGCTTGTGGGGAAGTCTC), complementary to a part of the 3'-untranslated region of the light chain (kappa), each sensitizer being located about 70 base pairs (bp) downstream of the end of the nucleotide sequence encoding the heavy chain domain lgM-CH1 of the transcript, or the constant region of the light chain kappa, respectively. In ia < to. < í £ -l «« -fom-i-l * ___. te___ * "_ -» «» - .. ».. 3¡¿ ^ **, ..-.« m -.-.- ^^? ^ ¡iij ^^ «ii ~: ^^^ faith ^ '^ ^ Both cases received nucleotide information from the Genebank database (http: // www ncbi.nim.nih.gov/htbin-post/Entrez/) for the kappa chain: Shepard and Gutman, Accession No. J02574, and for the mu chain: Parker, KE, Accession No. X68312 The poly-G tail using terminal transferase (Pharmacia, Freiburg, Germany) was then placed in the first cDNA strand according to the protocol common and current. The cDNA was amplified by PCR with tail, using a normal sense sensitizer containing a poly-C stretch, based on the anchor sensitizing sequence published by Gillliland, LK and coauthors (Tissue Antigens, 47, 1-20, 1996) and designated 5'-AncTail (CGTCGATGAGCTCTAGAATTCCCCCCCDCDCD). This anchor sensitizer was combined with a reverse sense sensitizer, specific for the nucleotide sequence encoding the C-terminus of the constant region of the light chain kappa, or the heavy chain domain lgM-CH1, respectively. The sensitizers were designated 3'-ratck: (GCGCCGTCTAGAATTAACACTCATTCCTGTTGAA) and 3'ratcmu (ATTGGGACTAGTCTCAACGACAGCTGGAAT). CPR was carried out in the following manner: Primary denaturation: 94 ° C for 4 minutes; 30 cycles of amplification: 93 ° C for 30 seconds; 55 ° C for 30 seconds; 72 ° C for 30 seconds; terminal lengthening: 72 ° C for 3 minutes. Each of these sensitizers contains a cleavage site of the restriction enzyme (5'-AncTail: EcoRI; 3'ratck: Xbal; 3'ratcmu: Spel), allowing the cloning of the corresponding PCR fragments in a | ___ ^ * .Jk & ._,., "« Aife vector plasmid digested with EcoRI / Xbal or EcoRI / Spel, respectively; for this purpose the plasmid vector Bluescppt KS + (Genebank, Accession No. X52327) was used, since it also allows for easy sequence analysis of the resulting inserts, using common sequence-forming sensitizers. Due to an Spel internal cleavage site within the variable region of the heavy chain (VH), partial digestion was necessary, followed by cloning of the full-length fragment, to obtain the complete sequence information of the VH domain. Partial digestion was carried out according to normal protocols (Sambrook, Cold Spring Harbor Laboratory Press, 1989, 2nd edition). Several clones of heavy and light chain fragments were shown to have identical sequences, respectively, and could be identified to encode VL functional regions or VH functional regions (see Figures 6 and 7). The mature N-terminus of both variable chains was identified by comparing their sequences with those found in the Genebank database (http: //www.ncbi nlm.nih.gov/htbin- post / Entrez /), and subsequently a second one was designed series of PCR sensitizers to introduce appropriate restriction enzyme cleavage sites, in frame with the coding sequences of the antibody fragment 2-B-5 Fab, and with respect to the requirements for subcloning into a bacterial expression vector. The two sensitizers were designated 5'RVHZXhol (CAGGTACAGCTGCTCGAGTCTGGGGCTGAGCTAG) and 5'RVJKZSacl (GTAAATGTGAGCTCCAGATGACACAGTCTCCTG) and used in combination with the 3'ratcmu and 3'ratck sensitizers, respectively. After PCR amplification and digestion with appropriate combinations of restriction enzyme 5 (Xhol / Spel for the heavy chain fragment and Sacl / Xbal for the light chain kappa) the resulting DNA fragments were cloned separately into the plasmid vector Bluescript correspondingly prepared and sequenced for confirmation (for the sequencing results see figures 6 and 7). For the expression of the fragment 2-B-5-Fab in the periplasm of E. coli, the corresponding cappa light chain of the Bluescript KS + was excised, using the restriction enzymes Sacl / Xbal and subcloned in the vector pComb3Hhis, prepared by digestion with the same enzymes. Then he digested the resulting plasmid with the Xhol / Nhel restriction enzymes and used as a vector to subclone the heavy chain Fd fragment 2-B-5 (VH + CH1); This DNA fragment of the corresponding Bluescript KS + clone was excised with the Hhol / Spel restriction enzymes. PComb3Hhis was derived from pComb3H and pComb3, respectively (Barbas and co-authors, Proc. Nati, Acad. Sci. USA 88, 7978-82 (1991)), by the following modification. The pComb3H vector was cleaved with Nhel and a double-stranded oligonucleotide was inserted by ligation, with suitable ends. HE created the double-filament oligomer by fixing the two A «* -.« ^ _ ^ ___ ^ _ * __ fl__, ______ a ^^^ l His6s 5'-phosphorylated sensitizers (CTAGCCATCACC ATCACCATCACA) and His (CTAGTGTGATGGTGATGGTGATGG) (at 94 ° C, 10 minutes, 65 ° C, 30 minutes, 52 ° C, 30 minutes and 30 ° C, 10 minutes). The sensitizer ends were designed so that, after fusion with the vector, the 3'Nhel restriction site was destroyed, while the 5'Nhel cleavage site remained intact. Finally, the insert was sequenced to confirm satisfactory cloning. Periplasm preparation was performed by osmotic shock and tested by ELISA for Fab fragments that bind to the zeta-peptide-KLH conjugate. For that purpose, individual colonies of E. coli XL1 ESlue, transformed with pComb3Hhis, containing the heavy chain Fd fragment and the light chain kappa of 2-B-5 in 10 ml of Super-Broth medium, supplemented with Carbenicillin and 20 mM MgCl 2 and Fab expression was induced after six hours, adding isopropyl-β-D-thiogalactoside (IPTG) to a final concentration of 1 mM. The cells were then harvested after 20 hours, redissolved in 1 ml of PBS. By four rounds of freezing at -70 ° C and thawing at 37 ° C, the outer membrane of the bacterium was destroyed and soluble periplasmic proteins, including Fab fragments, were released into the supernatant. After removing intact cells and cellular debris, by centrifugation, the supernatant containing the zeta-Fab-antibody fragment was collected, and used for further examination.

Jk? > ? ÁM.ÍL_itA __ *. t. KJtA ...... _. J,? _ *, __. T_, », ^." J &i, ¿¿¿¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡¡^ ^ ^ ^ ^ ^ ^ ^ ^ ^ The binding of the Fab fragment expressed periplasmically from the monoclonal antibody 2-B- was analyzed. 5 cloned, to the zeta-peptide-KLH conjugate, by the following ELISA. The antigen was immobilized in ELISA plates of 96 U-shaped concavities (Nunc Maxisorb) at a concentration of 200 μg / ml in 50 μl of phosphate-buffered saline (PBS) per concavity. The coating was carried out at 4 ° C for 12 hours, followed by a single washing step with PBS / 0.05% Tween. The ELISA was subsequently blocked for one hour with PBS / 3% bovine serum albumin (BSA) and rewashed. Then, preparations of periplasm containing Fab, undiluted and in various dilutions, were added and incubated for two hours. For the detection of Fab fragments attached to the zeta-peptide-KLH conjugate, a murine anti His-tag antibody (Dianova, Hamburg, Germany, Catalog No. DIA900) diluted 1: 200 was used, followed by an IgG antibody. goat anti-mouse, polyclonal, conjugated with peroxidase (specific for Fc-gamma) (Dianova / Jackson, Hamburg, Germany, Catalog No. 115-035-071), diluted 1: 5000. Finally the ELISA was revealed by adding ABTS-substrate solution (Boehringen, Mannheim, Mannheim, Germany, Catalog No. 1682008). The change of the colored substrate was measured by an ELISA reader at OD 405 nm; the results are shown in figure 5. As negative controls, concavities were incubated with PBS instead of periplasm preparations. Specific binding of several clones could be detected at é_.? _?.? : ^ ^^^^ SS¡ &Jji ^ conjugated zeta-peptide-KLH, with the results of clones 8 and 9 shown in Figure 5 Signal intensities varied distinctly over those of the negative controls, and he could titrate them with dilutions of the sample. The specificity for the zeta chain, of the cloned 2-B-5-Fab fragment, was confirmed.

EXAMPLE 7 STIMULATION OF T LYMPHOCYTES AND NK CELLS BY THE ANTI-BODY 2-B-5 ZETA ANTI-CHAIN The purpose of this experiment was the stimulation and proliferation of T cells, NK cells and PBMC induced by the anti-zeta 2-B-5 antibody. For this purpose, a colorimetric immunoassay was used, based on the measurement of incorporation of Bromodeoxyuridine (BrdU) during DNA synthesis (Boehringer Mannheim, Mannheim, Germany, Catalog No. 1647229). The first step in this analysis was to coat a 96-well flat-bottomed microtiter plate with the purified 2-B-5 antibody in several dilutions (for the purification of 2-B-5 see example 5). The coating was carried out overnight at 4 ° C. After washing three times with PBS, 100,000 CD8 + - T lymphocytes, NK cells and PBMC were added without separating, respectively, in triplicate, to the concavities of the titration microplate; CD8 + T-lymphocytes and NK cells were separated from _ .. t.i, + ",. **. ******* ** £ - *. z ¿¿^ ^^^^ & ^ & ^^ according to the instructions given in example 2, but using magnetic beads and the anti-CD8 (MT-811) and anti-primary antibodies -CD16 (3G8-lgG1 of ratio, Dianova, Hamburg, catalog No. 0813) respectively. To control the specificity of the stimulation mediated by 2-B-5, an antibody of the same isotype (rat IgM) with irrelevant specificity was used at the same concentrations. The IKT3 antibody (isotype lgG2a, Ortho, Product key 710320, Johnson &Johnson, New York, USA) was applied which recognizes the human CD3 complex, as a specific positive control, at a coating concentration of 1 μg / ml for the stimulation of T cells and PBMC without separating, respectively. A murine IgG2a antibody, with irrelevant specificity, was used as an isotype control for OKT3. A blank control (concavities without cells) and a background control (concavities without BrdU) were also included. After a three-day incubation period, the BrdU marker solution was added for 24 hours. During this marking or labeling period, the BrdU analogue to pyrimidine, instead of thymidine, is incorporated into the DNA of the proliferating cells. After removing the culture medium, the cells were fixed and the DNA denatured in one step, adding a denaturation solution. DNA denaturation is necessary to improve the accessibility of the incorporated BrdU, for detection by the anti-BrdU antibody, which is conjugated with peroxidase. This antibody binds BrdU incorporated in a cellular DNA, newly synthesized. The bound anti BrdU antibody was detected by the subsequent substrate reaction. The reaction product was quantified by an ELISA reader. The change of the colored substrate, when measured by absorbance values at a wavelength of 450 nm, correlates directly with the level of DNA synthesis and, thus, with the number of proliferating cells. All the steps were carried out as described in the equipment manufacturer's manual. The results of this analysis, as shown in Figure 8, clearly demonstrate that the 2-B-5 antibody not only binds to the short extracellular region of the zeta chain in T lymphocytes and in NK cells, but also induces strong stimulation of both types of cells, through this interaction.

EXAMPLE 8 CYTOMETRIC ANALYSIS OF FLOW OF THE INTERNALIZATION OF THE COMPLEX TCR / CD3, INDUCED BY THE UNION OF THE ZETA ANTI-CHAIN ANTIBODY For many receptors, activation by ligand binding is rapidly followed by internalization of the receptor. Accordingly, the rapid internalization of the TCR complex into T cells is typically observed after the binding of the anti-CD3 antibodies. Thus, the internalization of antibody 2-B-5 after binding to the TCR complex by means of its epitope of -t _ .. < jj -.- ..a-fa-t.-..-. . «-.su -»! ..-- go. ,? -. * - < , -. - ....._, and, _ * _ _ _____________ i ______ ± __ ___ 1 _________ u _ ^^ extracellular zeta chain, would confirm the peculiar specificity of the antibody of the invention (Boyer, C, Auphan, N, Luton, F., Malburet, JM , Barad, M, Bizozzero, JP, Reggio, H and Schmitt-Verhulst, AM (1991): T cell receptor / CD3 complex internalization following activation of a cytolytic T cell clone: evidence for a protein kinase C-independent staurosporine-sensitive step European Journal of Immunology, 21, 1623-34). In order to test the internalization of the receptor, after binding the antibody 2-B-5 to the surface of T cells, a flow cytometric analysis was carried out at different temperatures, which allowed the disappearance of the anti-cancer antibody to be observed. -Zeta chain attached to the surface. For this purpose, mononuclear cells were isolated from the peripheral blood of a healthy donor by centrifugation with a Ficoll density gradient. In each concavity of a titration microplate, 200,000 mononucleated cells were incubated with the anti-zeta 2-B-5 antibody at a concentration of 1 μg / ml, at 4 ° C or 37 ° C, for 30 or 60 minutes, giving enough time for the coronation to occur. A parallel experiment with a rat anti-human CD3 antibody (rat lgG2B) (clone 26-II 6-5) was carried out as a positive control. The coronation process was completed by washing twice with cold PBS. To label the antibody bound to the cell surface, the cells were incubated with a goat anti-rat antibody, fluorescein conjugate (FITC) (IgG + IgM) (Dianova / Jackson, Hamburg, Germany, No. catalog 112-016-044) diluted 1 100 in PBS. As a negative control, only the secondary antibody was used. The labeled or labeled cells were washed twice in PBS before fixing with PBS / 0.1% paraformaldehyde. The cells were analyzed by flow cytometry in a FACS scanner (Becton-Dickinson, Heidelberg, Germany). FACS spotting and fluorescence intensity measurement was performed as described in Current Protocols in Immunology (Coligan, Kruisbeek, Margulies, Shevach and Strober, Wiley-Interscience, 1992). As the results clearly show, an apparent displacement of the fluorescence intensity could be observed between the samples of cells incubated at 37 ° C, and those incubated at 4 ° C, after binding the anti-zeta 2B5 antibody. A similar internalization pattern could be observed after binding the anti-CD3 antibody. In contrast to internalization of the receptor at 37 ° C, samples incubated at 4 ° C, which is a non-permissive temperature for coronation events, revealed an unaltered fluorescence pattern.

EXAMPLE 9 CONSTRUCTION OF BIESPECÍFICO ANTIBODY BASED ON THE ZETA ANTI-CHAIN SPECIFICITY OF THE INVENTION To obtain a ScFv anti-zeta fragment, the «-ia» ..? * -, - »» «&» - £ t. -mfa i. < . «. ' «* .-. > ^. _. < _ .. jafe ^ -. ^ ¿«^^ t4ag ^^^^^^^« < ^^ g ^^^^^^^^^^^^^ | A ^^ I corresponding VL and VH regions, cloned in separate plasmid vectors, served as templates for a specific PCR for VL and VH, using the pairs of sensitizers of ohgonucleotide 5'VL2B5BsrGI-EcoRV / 3'VL2B5GS15 and 5'VH2B5GS 15 / 2'VH2B5BspEI, respectively. In this way, complementary overlapping sequences were introduced into the PCR products, which combine to form the coding sequence of a 3-linker of 15-amino acid (Gly4Ser1), during CPR by subsequent fusion. This amplification step was carried out with the sensitizing pair 5'VL2B5BsrGI-EcoRV / 3'VH2B5BspEI, and the resulting fusion product (or rather the scFv anti-zeta fragment) was cleaved with the restriction enzymes EcoRV and BspEl and, thus, it was cloned into a plasmid (described in PCT / EP98 / 07313), a preparation by digestion with the same restriction enzymes containing a scFv fragment, with specificity for binding against the EpCAM antigen, as well as a histidine tag. at the C end, for purification and analytical purposes. Subsequently, the DNA fragment encoding the single-chain, bispecific, anti-zeta / anti-EpCAM chain was subcloned with the dominance arrangement VLannZeta-VHant.Zeta-VHantiEpCAM-VLant.EpCAM, COI1 EcoRI / Sall, in the mammalian expression vector pEF-DHFR (Mack, M., Riethmüller, G and Kufer, P (1995): A small biospecific antibody construct expressed as a functional single-chain molecule with high tumor cell cytotoxicity. Proc. Nati, Acad. Sci USA 92, 7021-5). After the sequence confirmation (figure 10) the ^^ | and resulting plasmid DNA was transfected into CHO cells deficient in DHFR by electroporation; Selection was made for stable transfectants, gene amplification and protein production as described (Mack and coauthors). The bispecific antibody was purified by its h i st id i na label C-terminal, by means of affinity chromatography on a column Ni-NTA as described (Mack and co-authors). List of Sensitizers: 5'VL2B5BsrGI / EcoRV 5'-AAG TGT ACÁ CTC CGA TAT CCA GAT GAC ACÁ GTC TCC-3 '3'-VL2B5GS15 5'-GGA GCC GCC GCC GCC AGA ACC ACC ACC ACC TTT CAG CTC CAG CTT GGT CCC-3 '5'VH2B5GS15 5'-GGC GGC GGC GGC TCC GGT GGT GGT GGT TCT CAG GTA CAG CTG CAG CAA TCT GG-3"3'VH2B5BspEI 5" -AAT CCG GAA GAG ACÁ GTG ACC AGA GTG-3 ' EXAMPLE 10 CYTOTOXIC ACTIVITY OF PBMC AND CD8 + -LINFOCITS T, REDIRECTED AGAINST DESTINATION CELLS EpCAM-POSITIVES, THROUGH THE ZETA ANTI-CHAIN ANTI-CHAIN ANTIBODY / ANTIEpCAM In this experiment, target cells were marked with 51", Cr, washed, mixed with effector cells at a ratio of effector to target 20: 1 and subsequently incubated with different ^^^^^^^ í j k s ^^^^^^^ concentrations of bispecific anti-zeta-chain / anti-EpCAM. the amount of 51Cr released into the supernatant was quantified through the death of target cells and the lysis rate for each specific antibody concentration was calculated. For this analysis, human peripheral blood mononuclear cells (PBMC) or cytotoxic T lymphocytes, such as effector cells, were isolated from a fresh curd lining of a healthy donor. PBMCs were separated by centrifugation with Ficoll density gradient, followed by a subsequent centrifugation step (100 g) to remove thrombocytes. In order to isolate the cytotoxic T lymphocytes, a CD8 + subseries column kit (R & D Systems, Wiesbaden, Germany, Catalog No. HCD8C-1000) was used according to the manufacturer's protocols. 200,000 PBMC without stimulation or CD8 + T lymphocytes were added in a volume of 100 μl of RMPI 1640, medium supplemented with 10% FCS at each concavity of a round bottom microtiter plate, respectively. A cell line of gastric cancer cells, positive EpCAM, Kat III (ATCC HTB-103), labeled for one hour with chromium-51 (NEN-Life Science, Cologne, Germany, Catalog No. NEZ030S) was used as target cells. (with approximately 100 μCi); 10,000 target cells in a volume of 100 μl were added to each concavity of the microtiter plate. The bispecific antibody was added in concentrations of 40 ng / ml to 5 μg / ml in a volume of 50 μl. The microtiter plates were incubated during k4j__Jt_ |? _! _ j¡__íj __ ?? __ i______i___s_? ___ ^^^^^ 16 hours at 37 ° C, 5% of C02. At the end of the incubation period, 50 μl of supernatant was removed from each concavity and analyzed for the 51 Cr released in a gamma radiation counter (Wallac, 1480 Wizard 3", Freiburg, Germany). lysis of the target cells with a regulator containing Triton-X 100 (1.0% in PBS) The spontaneous 51 Cr release was determined by incubating the target cells without effector cells or bispecific antibody. with bispecific antibody did not result in measurable lysis.Specific lysis was calculated as follows: specific release (%) = [(cpm, experimental release) - (cpm, spontaneous release)] / [(cpm, maximum release) - (cpm, spontaneous release)] x 100. All tests were carried out in triplicate, the standard deviation within the triplicates was less than 6% in all experiments. the purity of isolated CD8 + effector cells, by flow cytometry; NK cell contaminations were excluded by staining with an anti-human CD56 antibody, conjugated with PE. The FACS analysis was performed as described in example 1. The following antibody conjugates were used: anti-human FITC-CD3 (Pharmingen, catalog No. 30104X) 1:40; Anti-human PE-CD56 (Becton Dickinson, Catalog No. 347747) 1:20; Tricolor mouse anti-human CD8 (Caltac Lab, code No. MHCD0806) 1: 100 (Becton Dickinson). The results of the FACS analyzes confirmed the high purity of the ., _.r _,? Ii _ _?? _ TA «&t -. < - ^^^^^ Llt ^^^^^^^ population of CD8 + cells (> 99%). The results (FIG. 11) of the chromium release assay clearly demonstrated the ability of the bispecific anti-zeta / anti-EpCAM antibody to redirect the cytotoxic T lymphocytes of PBMC or CD8 + without stimulation, against the KATO III EpCAM-positive cells. The differences between the specific lysis mediated by non-separated PBMC and isolated cytotoxic T lymphocytes are explained by the cytotoxic contribution of NK cells.

EXAMPLE 11 CYTOTOXIC ACTIVITY OF < NK CELLS REDIRECTED AGAINST DESTINY CELLS ED CAM- POSITIVE, THROUGH THE ANTI-CHANNEL ANTIBODY ANTI-CHANNEL ZETA / ANTI EpCAM This experiment was designed to demonstrate the ability of the bispecific anti-zeta / anti-EpCAM antibody to redirect NK cells against EpCAM-positive target cells. To perform this analysis, NK cells were isolated from human peripheral blood mononuclear cells (PBMC), using an NK cell isolation kit (Militenyi Biotec, Bergisch Gladbach, Germany, Order No. 465-02). The isolation strategy is based on magnetic depletion of non-NK cells. T cells, B cells, monocytes, basophils, dendritic cells, and platelets are indirectly labeled using a combination of hapten-conjugated antibodies CD3, CD14, CD19, CD36, and anti- ... ÍA t -,. ", *.: .., *, * ..., *. .J ». - -. ^ -. i .. «> * tft ^^ ¡^ ~ ~ IgE, followed by paramagnetic beads coupled to an anti-hapten monoclonal antibody. The cells associated with the magnetic beads were retained by virtue of a magnetic field. The NK cells were washed without marking through the column and remained untouched. 100,000 NK cells were added from each of three healthy, different donors in a volume of 100 μl of RPMI 1640 medium supplemented with 10% FCS to each concavity of a microtitre plate, round bottom, respectively. As a target, marked Kato cells were added to each concavity with 51 Cr (10,000 target cells per concavity, in a volume of 100 μl each). Bispecific antibody was added at a concentration of 1 μg / ml in a volume of 50 μl. The microtitrator plates were incubated for 4 hours at 37 ° C, 5% C02. At the end of the incubation period 50 μl of supernatant was removed and was analyzed for the 51Cr released in a gamma counter (Wallac, 1480 Wizard 3", Freiburg, Germany). The maximum release of 51 Cr was determined by lysis of target cells with a regulator containing a detergent (1.0% Triton). X 100 in PBS) The spontaneous 51 Cr release was determined by cell incubation target without effector cells or bispecific antibody. Incubation of target cells with bispecific antibody alone did not result in measurable lysis. The specific lisis was calculated as follows: specific release (%) = [(cpm, experimental release) - (cpm, spontaneous release)] / [(cpm, release maximum) - (cpm, spontaneous release)] x 100. It was carried out jfaa; Ay. «s, J--« i ^ .... ... .. ... i. . i _._._- _-__ ^ ___ ¿__i _.____. all tests in triplicate; the standard deviation within the triplicates was less than 6% in all experiments. The purity of the isolated NK effector cells was analyzed by flow cytometry; Contamination with CD8 + cells was excluded by staining with a mouse anti-human CD8 antibody, conjugated with tricolor. The FACS analysis was carried out as described in Example 1. The following antibody conjugates were used: anti-human FITC-CD3 (Pharmingen, catalog No. 30104X) 1:50; Anti-human PE-CD56 (Becton Dickinson, Catalog No. 347747) 1:20; Tricolor-CD8 anti-human mouse (Caltac Lab, code No. MHCD0806) 1: 100 (Becton Dickinson). The results of the FACS analyzes confirmed the high purity of the NK cell population (> 98%). The results (Figure 12) of the chromium release analysis demonstrated in all three cases a reproducible ability to redirect NK cells against EpCAM-positive target cells. i.-J.d ^ - ^ -.- i. -ÜJ-É-á-, LIST OF SEQUENCES < 110 > Connex GmbH < 120 > Immunological reagent that interacts specifically with the extracellular domain of the human zeta chain < 130 > C1358PCT < 140 > < 141 > < 150 > EP 98 11 2867 1 < 151 > 1998-07-10 < 160 > 18 < 170 > Patentln Ver. 2 1 < 210 > 1 < 211 > 33 < 212 > DNA < 213 > Rattus norvegicus < 220 > < 221 > CDS < 222 > (1) ... (33) < 400 > 1 cag gca age cag gac att ggt aat tgg tta gca 33 Gln Ala Ser Gln Asp lie and Asn Trp Leu Ala 1 5 10 < 210 > 2 < 211 > 11 < 212 > PRT < 213 > Rattus norvegicus < 400 > 2 Gln Ala Ser Gln Asp lie Gly Asn Trp Leu Ala 1 5 10 < 210 > 3 < 211 > 21 < 212 > DNA < 213 > Rattus norvegicus < 220 > < 221 > CDS < 222 > (1) ... (21) < 400 > 3 agt gca acc age ttg gca gac 21 Ser Ala Thr Ser Leu Ala Asp 1 5 < 210 > 4 < 211 > 7 < 212 > PRT < 213 > Rattus norvegicus < 400 > 4 Ser Ala Thr Ser Leu Ala Asp 1 5 < 210 > 5 < 211 > 27 ssaüas ^ - .í £ f < 212 > DNA < 213 > Rattus norvegicus < 220 > < 221 > CDS < 222 > (1) -. (27) < 400 > 5 cta cag cgt tat agt aat ecc aac acg 27 Leu Gln Arg Tyr Ser Asn Pro Asn Thr 1 5 < 210 > 6 < 211 > 9 < 212 > PRT < 213 > Rattus norvegicus < 400 > 6 Leu Gln Arg Tyr Ser Asn Pro Asn Thr 1 5 < 210 > 7 < 211 > 30 < 212 > DNA < 213 > Rattus norvegicus < 220 > < 221 > CDS < 222 > (1) ... (30) < 400 > 7 faU lA .- .. * -. * MJa l ".íí_Éí_, i.« _. ik -i «.i-.»,? Jib &9dk »? S¿fc ____ t______s__ ggc tac here ttc acc agt tac gat atg cac 30 Gly Tyr Thr Phe Thr Ser Tyr Asp let His 1 5 10 < 210 > 8 < 211 > 10 < 212 > PRT < 213 > Rattus norvegicus < 400 > 8 Gly Tyr Thr Phe Thr Ser Tyr Asp Met His 1 5 10 < 210 > 9 < 211 > 51 < 212 > DNA < 213 > Rattus norvegicus < 220 > < 221 > CDS < 222 > (1) ... (51) < 400 > 9 ttg att tat cct gga aat ggt aat act aag tac aat ca aag ttc 45 Trp lie Tyr Pro Gly Asn Gly Asn Thr Lys Tyr Asn G n Lys Phe 1 5 10 15 aat ggg 51 Asn Gly < 210 > 10 < 211 > 17 < 212 > PRT < 213 > Rattus norvegicus < 400 > 10 Trp lie Tyr Pro Gly Asn Gly Asn Thr Lys Tyr Asn Gln Lys Phe Asn 1 5 10 15 Gly < 210 > 11 < 211 > 42 < 212 > DNA < 213 > Rattus norvegicus < 220 > < 221 > CDS < 222 > (1) (42) < 400 > 11 gat tgg cat tac tat age age tat ate cgt ecc ttt gct tac 42 Asp Trp His Tyr Tyr Ser Ser Tyr lie Arg Pro Phe Ala Tyr 1 5 10 < 210 > 12 < 211 > 14 < 212 > PRT < 213 > Rattus norvegicus < 400 > 12 Asp Trp His Tyr Tyr Ser Ser Tyr lie Arg Pro Phe Ala Tyr 1 5 10 .JL *. . * «.--., .-» -. ~ - ~. * - -. - * ~ ¿_ ^ -_-. ^ _____ s____ _ _____j < 210 > 13 < 211 > 369 < 212 > DNA < 213 > Rattus norvegicus < 220 > < 221 > CDS < 222 > (1) ... (369) < 400 > 13 cag gta cag ctg cag caa tet ggg gct gaa cta gtg aag cct ggg tcc 4í Gln Val Gln Leu £ 1A Ctfn? -ír Gly? The Glu Leu Val Lya Pro Gly Ser 1 5 10 15 tea Qt aa att tcc tgc aag get tet ggc téi. here ttc acc agt tac 96 Ser Val Ly $ lie Se C fc Ly_. Wing 01 v Tvr Tbr Phe Til i "Ser Tyr 20 25 30 g &t atg falls tgg ata aaa cag Cftg cct gga aat ggc ctt gag tgg atí- 144 Asp Ket Kis Trp lys LyS G r; Gln Pro ly ' Agn Gly eu <Lü 'Trp Ele 35 40 45 15 gs? R tgg att tst cct gga aat gsc aat ac aag tac aa-L ca aag tte 192 Gly Trp? Le? Yr P o Cly ASE Gly A n n Thr Lye Tyr Asn ISL? Phe 50 55 60 aat ^ asg gca here ctc ect gca aac aaa tc age aac gcc ta 240 Aen Gly Lye Ala Thr Leu Thr Ala Ae_> Lye Ser Ser Ser T Ala Tyr 65 • 70 .75 30 aey cag ctc age age ctg here tet gas Gac ect gc gtc tat ttc tgt 2S8 iíet Glr¡ Leu Ser Ser u T? go S? r Glu Asp Ser? the Val Tyr?:? < = Cy £ &S 30 $ 5 20 gca aga gac cgg eat tac tat are age tac ate critee t.tt gct tac 336- Wing Arg Asp Trp Hxs' XVr Tyr Ser Ser Tyt lie? Rg Prc Phe Wing IVr 100 105 110 tgg ggc ca ggc aet ctg gtc act gtc tcc tea 369 Trp Gly Gln Gly Tl = r Leu V l 71.? Val Ser Ser 115 120 < 210 > 14 < 211 > 123 < 212 > PRT < 213 > Rattus norvegicus < 400 > 14 Glp Val (51 n Lfeu Gla Glr; Ser Gly? Glu Leu VaL Ly-S PZ? Gly Ser 1 5 L 15 Ser Val Lys laugh Ser C &Lvs Wing Ser Gly Tyr ih Phe Tir Ser T 20 S JO Asp Met His Trp? Le Ly * Gln Gll Pro Gly ASp Gly Leu Glu Trp? 40 < -; • Giy Tr? Lie Tyr Ere Gly Asn £ l and so r Ly-5 Tyr A &p GIG? Lye? Be 50 55 60 Asn Gly L s Wing Thr Leu Thr Wing Aep Lye Ser Ser. Ser T? Go Wing Tyr 65 70 75 80 Mee Glp Leu $ er $ - ri »cu Tht Ser Glu Asp Ser Wing Val Tyr P? Ie Cy 90 95 Wing Arg Aep Trp Bis Tyr Tyr Ser Se Tyr lie Arg Prc Phe Wing Tyr 10 105 U0 Tro Gly Gln Glv Thr Leu Val Thr < ral sr Ser 115"20 < 210 > 15 < 211 > 321 < 212 > DNA < 213 > Rattus norvegicus < 220 > < 221 > CDS < 222 > (1) ... (321) < 400 > fifteen gact ate cag atg ac cag tet ect gct tcc ctg t.t Seg tet ceg gaa 48 As ríe í. Wet Tht- Gln? € Pro Ala Ser Leu 3rd Wing Ser Pro Glu 1 5 10 15 gaa att gtc acg ate ac tac cag gca age cag gac att ggt aat tgg Glu He val Thr l rfcr Cye Gn Ala Ser Gn Aep lie Gy «Sa Trjp 20 25 tta gca tg tat cag cag ea.5. cca e aaa tet cct coa ctc ctc at 144 Leu Ala Trp Tyr slñ Gla Lys Pro Gly Lys Ser Pro G GJ Leu Leu 11 ß 35 AO i5 tat gt í? ca acc aeje ttg g e gac ggg ate ce? ttc age ggc ± $ 2 Tyr Ser Ala Thr £ e e Ala? YES. Gly Ile Fro Ser Arg Phe Ser C? Y 50 55 Gil agt aga te gt ac cag tat tet Ctt aag ate acc aga cta cag Qtt 2AQ Ser Arg 3rd Gly Thr Gln Tyr s r L.v. Lys I Ser Arg L u Gln v l • 55 7 75 60 10 sraa .aat acc ate tat tag tgt cta cag cgt tac agt aat Cúc aac 2ßs Glu Aep Thr Gly lie T_'r- Tyr ys < SU Gla Arg l r Ser Á? * ro A?,:? 50 25 acg ttt ega set geg acc aag ctg gag etg aaa 221 Thr Phe Gly Wing Gly? Br Lyg Iie? Glu ey Lys 100 105 < 210 > 16 < 211 > 107 < 212 > PRT < 213 > Rattus norvegicus < 400 > 16 J ^ ¡^ ^^^^^^ - ^^^^^^ _ ^ _ ^^ _ ^^^^^. "- j í. * * Aep He C- n Met Thr s n 3rd Pro Wing S? Leu Be Ala Be Pro Glu 1 5 10 15 Glu ie Val Thr lie- Thr Cys 01n Ala Se Glfl AS? > ie Glv Asn Trp 20 25 30 LffU Wing Trp Tyr Gln Gln Lya ro Gly Lys = er Pro Gll Leu Le He 35 45 Tyr Ser Ala T r Ser Leu Ala Aep Gly lio Pro Ser A g £ > he $ er G? y 50 55 60 Ser Arg = er C-ly T-go < 31n Tyr 3er eu ya lió Ser Arg Leu Gin Val 65 70 75 T0 Giu- Asp Thr Gly He Tyr Tyr Cys __, e? Gln Arg Tyr-Sar Aén Ero Aan = 5 $ 0 95 Thr? B. < = Gly Al Gly Thr Lya Leu Glv Lya - 100 105 < 210 > 17 < 211 > 1637 < 212 > DNA < 213 > Artificial sequence < 220 > < 223 > Description of artificial sequence: artificial sequence. < 400 > 17 • i í SST S-assss t.acagíftgta SC ^ r t aag aa 120 gtatcagcsg 1 &0 agacgggatc Iu cag agacta caacacgttt 300 360 420 cggctccggt gcctgggtcc 480 is tgg agc 540 ataaaa egcctggaaa tgcce CGIA tggattgg GGAT t tg t-Etcc aaatg- gt GOO aa actaagt scaaccaaaa gi caatggg aaggeaEcac í-.cp.-ct ca caaatcc cc 660 agcacagcct atatgeaget cagcagcctg acate gagg actccacagt. ctat tc gt 720 gcaagagatt ggeattact tag agerat a cegtecet t c tactg ggg csagec 7SO ac c ggtce ctgtctc tc c e gi.3Cg i; ggttetgagg tgcagc gt cgagca tet 54 ggag tgagc ggcgaggce tg-gg-ge ea gtgEagctgt cc gcadg r tctg c ac 900 accttcacaa actat gt t aagctggg g aagcagaggc ctggacaggt c tg? gtgg 960 tttatcctag eatt gdaac g ctactaca atga aagtt caaggg aag 1UÜÜ gccacactge ctgeagacaa atcctccs c acaacgtcca tggagctccg catrcctgae tc gagg ct ccgcggtcta tttctg ca agacggggat cctacgstac a tSegac 2140 tg ta tcß &CQ'tCtggg.g ccaa gEcc -acggccaccg tctcctcagü tggtggtggt í ttig grj gcggct cgg tggt-ggtggt t gagcccg tgat accca actccactc 2? .60 tcCctgcctg tcagtettgg agatcaaücc tCC ?? tCtCtt gcagatctag tcr-gag ctt. 1320 gtaca? Agte atggaaacac etatttacat tggtacctgc agaagecagg ccaatc cca 13B0 sag tcctga cc ".asa agt ttccaaccga tttt-. Tgggg tcccagaca? I gttcag ggc 1-J40 agcgg e u ggacagatt cacactceag ateageagag ggatctggga 3.S0O Sttt-? I.tttc gc c caaag tacacaeett cc tacac t tcggaggggg gaccaagccc 1560 gagatcaaac gta gactag ecatcaocat accatcaca etegeteatt aattcaa ^ cg 1520 eccgctctaf. a tC'írac 1S37 < 210 > 18 < 211 > 532 < 212 > PRT < 213 > Artificial sequence < 220 > < 223: > Artificial sequence artificial sequence description < 400 > 18 Ifé- Gly TrJ > Ser Cys lie He eu Pb © &? Val Ala Thr Ala Thr Gly 1 5 10 15 Val Kls 5c As lie Gln Me T r Gln = er Pro? La Ser Leu Ser Ala 2. Z $ 30 Ser Pro Glu Glu H Val Thr lie 2h Cys Gln Ala-Se: R Glr) ASy He 35 4Ü Í5 Gly Asn Trp Wing XO > Tyr G n Gln Lya PrO-Gly Lya Ser _? or Gln I ^^^^ J ^? J ^ »^ -JkÉJ > * > ----..- 50 55 GO L «¡.OU lis Tyr Ser Ala; - Be ev¡ Ala? Sp G? And ILe Pro Se Axg 65 7lj 75 30 Ph Ser Giy Ser r ^ Ser Glv Ekr G-ln Tyr be JCeu Lys laughs Se Arg -35 $ 0 95 Leu ír. Val Glu A-SP Thr Gly He Tyr Tyr Cys u Gln Arg T r Ser IDO IOS 110 5 Asn P-ro Asn Thr Phe Gl? La Gly T 'rs L? Tl Glu Leu Lys Gly Cl 115 120 125 Gly Gly S? r Gly Gly Gly Gly Ser C-ly Gly Glv Gl S Glp al Gln 130 135 140 Le Glr. G n Ser Gly Wing Glu Leu Val __y =. ? ro Gly Ser Ser Val Lvs 145 150 155 l = O lie Ser Cys Lys Ale Ser Gly 1Yr Tt: r Phe Tbr Ser Tyr Ase Me Hls 165 17C 175 1n Tp He Ly = Glp. Gln Pro Gly f.sn Gly eu < 3ltt Tr lie Gly T'rp lie , u? ao í is iso Ty Pro Gly Asn Gly Afir. Tiiir Lya Tyr Aan Gln Lys Phe Asn Gly yfe 155 200 2-05 Wing Thr Leu Thr Ale AS »L a S? R Ser Ser Thr Ala Tyr 1-íet C-ln Leu 210 215 220 Ser Ser Leu Thr Ser Glu Asp sr Wing Val Tyr Pbe Cys Wing Arg Asp 225 23-0 235 2 0 T? P Kis- Ty Tyr Ser Ser Tyr lie Arg Pro * Ala Wing Tyr Tr Glv Gln 245 250 255 15 Gly Thr Leu at Thr val Ser Sat Gly Gly Gly Gly Ser Glu Val Gln 260 265 270 Leu Leu Glu Gln Sar Gly Wing Glu Leu A a Ar = r Pro Glv Wing Ser vl 275 2S0 235 LVS Z. XI. Ser Cys Lys Wing Ser Gly 'jyr rhr? e Thr ASM Ty Gly Leu 90 295 300 $ e Trp See! Uys Gln Pro Gly Glp Val Leu Glu Tre lie Gly Glu 305 310 315. 320 Val Tyr? Ro Ar He Gly Asn Ala see Tyr Asp Glu Lys Phe Lys Glv 325 330 '355 Lys Wing Thr Leu rhr Wing ASP L s Be Ser Thr Wing Ser Mee Glu 3Ú0 345 350 Leu -via Ser Leu Thr Ser Glu Asp Ser Wing Val Tyr Phe Cys Wing Arg 355 350 365 Arg Gly Ser Tyr Aso Thr ASn Tvr Asp T'rp Tyr Plie As Val Trp GLy 370 375 - - 380 C-lp Gly T rcr Val Thr Val Ser Ser Gly Gly Gly Gly Ser í? L GLy 39S 390 395- 400 - ^^ ggg? | »jg¡g ^^^^^^^^^ ..... ^^^^^^^^^^^^^^^^^^^^^^^^^^ Gly Gly & Gly Gly Gly Ser Glv, Leu Val Met Thr Gln. Rjas Pro 405 410 415 Leu Ser Leu P or Val S r Leu Glv Aap Qln .Ma e < ? r He- 3er goes Arg 420 Ser Ser G p. Ser Leu £ l His Ser Aan Gly Aso T-v Tyr Leu Hie, Trp 43S i-10 445 Le? Gin Lve Pro Gly G Ssr Pro Lys Leu Leu Laughs Tyr Lye Val 450 55 «5Q Ser Aen? Plie Se Gly Val Fr? ? s Arg Phe Ser Gly = <; r Gly S < = z-4 < ? 5 ¿70 475 ÍS0 Gly Thr Aap? < s? br Leu Lys lie • Ser Arg Val Glu A ?? Glu Asx? Le 4S5 490 $ 5 Gly Val Tyr Phe C a Se < 3in Be Thr Hie Val? Tyr -Thr Ppe Glv 50Ú SOS 510 Gl Ü and Th Le C-lu? Le Lys Arg Thr Thr Ser Ei: Ls íiis Kiá His 515 Hie Kie Thr $ p- 520 tM * ._ ^ Jt «fc. Úit. U ^ j ^ ¡j¡¡

Claims (38)

1. - A nucleic acid molecule, characterized in that it comprises a nucleic acid sequence encoding at least one complementary determining region (CDR) of a variable region of an antibody; wherein said at least one CDR, alone or in combination with at least one more CDR, is sufficient to contribute at least to a weak, but significant, binding of the antibody to the extracellular domain of the human zeta chain; said antibody being obtainable by immunizing a rat with Jurkat cells and subsequently with a conjugate comprising a carrier molecule and a peptide comprising the 11 N-terminal amino acids of the rat zeta chain.

2. The nucleic acid molecule according to claim 1, further characterized in that the nucleic acid molecule comprises a nucleic acid sequence encoding at least two CDRs of the variable region.

3. The nucleic acid molecule according to claim 1 or 2, further characterized in that the nucleic acid molecule comprises a nucleic acid sequence encoding three CDRs of the variable region.

4. The nucleic acid molecule according to any of claims 1 to 3, further characterized in that the nucleic acid sequence encodes a VH chain.

5.- The nucleic acid molecule in accordance with

> < ttt ^. ^ ¿~ T * ... t »»,. ^. ^ ^ y- »----- jfffrff any of claims 1 to 3, further characterized in that the nucleic acid sequence encodes a VL chain.

6. The nucleic acid molecule according to any of claims 1 to 5, further characterized in that it is a DNA molecule.

7. A nucleic acid molecule, characterized in that it comprises a nucleic acid sequence encoding at least two CDRs of a variable region of a VH chain; said antibody specifically interacting with the extracellular domain of the human zeta chain; said antibody being obtained by immunizing a rat with Jurkat cells and subsequently with a conjugate comprising a carrier molecule and a peptide comprising the eleven N-terminal amino acids of the rat zeta chain.

8. A nucleic acid molecule, characterized in that it comprises a nucleic acid sequence encoding at least two CDRs of a variable region of a VL chain; said antibody specifically interacting with the extracellular domain of the human zeta chain; the antibody being obtainable by immunizing a rat with Jurkat cells and subsequently with a conjugate comprising a carrier molecule and a peptide comprising the eleven N-terminal amino acids of the rat zeta chain.

9. The nucleic acid molecule according to any of claims 1 to 8, further characterized in that the CDR has one of the following nucleotide sequences: (a) SEQ ID NO. 1 (b) SEQ ID NO. 3 (c) SEQ ID NO. 5 (d) SEQ ID NO. 7 (e) SEQ ID NO. 9 (f) SEQ ID NO. 11. The nucleic acid molecule according to claim 4, further characterized in that the VH chain has the nucleotide sequence of SEQ ID NO. 13 or encodes the amino acid sequence of SEQ ID NO. 14. The nucleic acid molecule according to claim 5, further characterized in that the VL chain has the nucleotide sequence of SEQ ID NO. Or encodes the amino acid sequence of SEQ ID NO. 16. The nucleic acid molecule according to claim 7, further characterized in that the VH chain has the nucleotide sequence of SEQ ID NO. 13 or encodes the amino acid sequence of SEQ ID NO. 14. The nucleic acid molecule according to claim 7, further characterized in that the V chain has the nucleotide sequence of SEQ ID NO. Or encodes the amino acid sequence of SEQ ID NO. 16. 14. The nucleic acid molecule according to any of claims 1 to 8, further characterized in that the CDR encodes one of the amino acid sequences: (a) SEQ ID NO. 2 (b) SEQ ID NO. 4 (c) SEQ ID NO. 6 (d) SEQ ID NO. 8 (e) SEQ ID NO. 10 (f) SEQ ID NO. 12. A vector, characterized in that it comprises the nucleic acid molecule of any of claims 1 to 14. 16. A host transformed or transfected with the vector of claim 15. 17. A method for producing a ( poly) peptide encoded by the nucleic acid molecule of any of claims 1 to 14, characterized in that said method comprises culturing the host of claim 16 under suitable conditions, and isolating the (poly) peptide from the culture. 18. A (poly) peptide, characterized in that it is encoded by the nucleic acid molecule of any of claims 1 to 14, or produced by the method of claim 17. 19. An antibody or a fragment or a derivative of it, characterized in that they comprise at least one (poly) peptide of claim 18. 20. The antibody according to claim

19, further characterized in that it is a monoclonal antibody. 21. The antibody according to claim 19, further characterized in that it is a bispecific antibody. 22. The antibody according to claim 21, further characterized in that the first specificity is for the extracellular domain of the human zeta chain on the surface of an intact cell, and the second specificity is for an optionally different molecule, on the surface of a T lymphocyte, a natural killer cell or a precursor of them. 23.- The antibody in accordance with the claim

21, further characterized in that the first specificity is for the extracellular domain of the human zeta chain on the surface of an intact cell, and the second specificity is for a different molecule on the surface of a different cell. 24.- The antibody in accordance with the claim

23, further characterized in that the different cell is a different cell, selected from a T cell, an NK cell or a precursor thereof. 25. The antibody according to claim 23 or 24, further characterized in that the different molecule is an antigen encoded by virus, an antigen associated with tumor or a surface antigen, any of them in cells that present antigen (APC) or in non-APC cells. 26. The antibody according to claim 25, further characterized in that the APC is a dendritic cell.

27. - The derivative according to claim 19, further characterized in that it is a scFv chain. 28. The antibody according to claim 20, further characterized in that it is an IgM. 5 29. A bispecific receptor characterized in that it comprises a (poly) peptide of the claim and a natural receptor, a natural ligand or derivatives thereof, which interact with a surface molecule in the same cell or in another cell. 30. The bispecific receptor according to claim 10, further characterized in that the receptors or ligands are CD4, CTLA-4, B7-1, B7-2, LFA-3, ICAM-1, 2, 3 or chemokines as MIP1a, MIP-1B, RANTES or SDF-1. 31. A pharmaceutical composition, characterized in that it comprises the nucleic acid molecule of any of claims 1 to 14, the vector of claim 15, the host of claim 16, the (poly) peptide of claim 18, antibody or its fragment or derivative of any of claims 19 to 28 and / or the bispecific receptor of claim 29 or claim 30. The use of the antibody according to claim 22, for the preparation of a pharmaceutical composition for the treatment or prevention of autoimmune diseases, immunological deficiencies, T cell malignancies, infectious diseases or for the suppression of the immunological response.

^^^^ ¡¡¡¡¡¡¡¡-!,. 4 i fc ^ a »fc-¿a

33. The use according to claim 32, further characterized in that the suppression of the immune response is to be carried out in order to avoid rejection of the graft after an organ transplantation. The use of the antibody according to claim 23, for the preparation of a pharmaceutical composition for the treatment or prevention of malignancies, viral infections or other infectious diseases. The use of the (po li) peptide of claim 18 or its antibody or its fragment or derivative of any of claims 19 to 28, or the bispecific receptor of claim 29 or 30, for the preparation of a pharmaceutical composition for the increase or suppression of immunity that depends on the NK cell, or for the treatment of malignancies derived from the NK cell. 36.- A method for the determination of the expression of chain zeta or chain eta in NK cells, T lymphocytes or their precursors, characterized in that it comprises: (a) contacting the (poly) peptide of claim 18 or 20 antibody or its fragment or derivative of any of claims 19 to 28, with said NK cells, T lymphocytes or their precursors; and (b) determining the amount of bound (poly) peptide, antibody or derivative. 25 37.- A kit or case characterized in that it comprises

2 ^^ á_W__É ____________ _______ ^^ «----« .. -i -.--- * _ & «. - i ^ i-i-j ia-jALI, the nucleic acid molecule of any of claims 1 to 14, the vector of claim 15, the host of claim 14, the (poly) The peptide of claim 18, the antibody or its fragment or derivative of any of claims 19 to 28 and / or the bispecific receptor of claim 29 or 30. 38.- A non-human transgenic animal, characterized in that it comprises in its line germinating at least one copy of the nucleic acid molecule of any one of claims 1 to

10 14 or the vector of claim 15.