MXPA00000643A - Directed cytolysis of target cells, agents and compositions causing cytolysis, and compounds that can be used to produce the agents - Google Patents

Abstract

A method for inactivating target cells in the presence of T cells by bringing the two types of cells in contact with a superantigen (SAG) in the presence of an immune modulator, characterized in that at least one of the superantigen and the immune modulator is in the form of a conjugate between a"free"superantigen (Sag) and a moiety targeting the conjugate to the target cells. A superantigen conjugate complying with the formula (I):(T)x(Sag)y(IM)z;a) T is a targeting moiety, Sag corresponds to a free superantigen, IM is an immune modulator that is not a superantigen and T, Sag and IM are linked together via organic linkers B;b) x, y and z are integers that typically are selected among 0-10 and represent the number of moieties T, Sag and IM, respetively, in a given conjugate molecule, with the provision that y>0 and also one or both of x and z>0. The superantigen conjugate is preferably a triple fusion protein. A targeted immune modulator, characterized in that it is a conjugate between a targeting moiety (T''') and a modified immune modulator (IM'''). The conjugate complies with a formula analogous to formula (I) except for the imperative presence of the modified immune modulator. A superantigen moiety may be present. A DNA molecule encoding a superantigen and an immune modulator.

Description

CITOLYSIS DIRECTED TO OBJECTIVE CELLS, AGENTS AND COMPOSITIONS THAT CAUSE CYTOLYSIS, AND COMPOUNDS THAT CAN BE USED TO PRODUCE THE AGENTS.

Field of the Invention The present invention relates to the inactivation / cytolysis of target cells caused by T cells that are activated by functional superantigens. Cytolysis can be applied to therapy and in vitro assessment.

Definitions Superantigens. According to the first definition (around 1988-1993), superantigens are bacterial or viral proteins with the ability to bind MHC class II antigens without the need for prior intracellular processing and activate T cells by binding in the variable region of the chain -Vß (Vß) of the T cell receptor (TCR). The binding leads to a restricted activation of the Vß family of a relatively large proportion / subset of T cells and a lysis of the cells expressing REF: 32563 for the MHC class II (cytolysis mediated by a cell-dependent superantigen = SDCC ) Typically, the subset of T cells activated by a superantigen constitutes approximately 1-30% of the total amount of an individual's T cells.

According to the above definition, the wild type superantigens that are well known are the staphylococcal enterotoxins (SEA, SEB, SEC1, SEC2, SED SEE and SEH). In addition, other examples are Toxin I of Toxic Shock Syndrome (TSST-1, also of staphylococcal origin), Exfoliating Toxins (EXft), Streptococcal Exotoxin of Streptococcus A, B and C (SPE A, B and C). , from Mouse Mammary Tumor Virus Proteins (MMTV), of Streptococcal M Proteins, from the Enterotoxin of Clostridium um perfringens (CPET), of superantigens of mycoplasma arthritis, etc. for a review of superantigens and their properties see Kotzin et al 1993.

The chimeric and wild type superantigens have also been mutated to have a reduced binding to MHC class II or not and / or a TCRVβ binding (Kappler et al WO 931264 / Kapler et al 1993; Blanco et al; Abrahmsén et al., WO9601650; Antonsson et al W09736932; Antonnson et al. 1997). This type of superantigens become less toxic. In case they are completely lacking the ability to bind MHC class II or TCRVß they are no longer functional superantigens because then they lose the ability to activate the T cell.

By mutating the structurally similar wild-type superantigens, it becomes possible to construct functionally active chimeric superantigens (hybrid superantigens) (Lamphaer et al., 1996 and Antonsson et al WO 9736932).

It has been discovered that activation and subsequent cell lysis can occur independently in a class II MHC in case the wild-type superantigen has been conjugated with a target-seeking portion capable of binding to the structure of the cell surface (Dohlsten et al WO9201470). This new effector mechanism has been called cell-dependent antibody superantigen-mediated cytolysis (= SADCC). This includes analogous mechanisms for targeting portions other than antibodies (Abrahmsen et al., WO9601650, Antonsson et al WO 9736932).

Accordingly, the current superantigen concept encompasses any compound (preferably of a polypeptide structure) that without intracellular processing is capable of binding to the cell surface structure (target structure) and to one or more polymorphic TCR chains, in particular to the chain Vß, and therefore activates a subset of T cells expressing for the specific TCR chain involved in the binding. The T cells then become cytotoxic and direct their cytotoxicity against the cells that carry the surface structure (objective structures, target cells). The definition of superantigen (SAG) as used in the context of the invention, and if not specified otherwise, will thus encompass conjugates between a targeting portion and an independent superantigen as discussed above for the SADCC.

By the term superantigen, it is contemplated, if not otherwise specified, only to functional superantigens.

An independent superantigen (Sag) is a wild-type superantigen, which has possibly mutated or otherwise modified, which is not conjugated to a targeting or immunomodulating portion. The binding ability of MHC class II to independent superantigens is an inherent property to go to the target. Since independent superantigens lack conjugated conjugate portions, they only exert SDCC.

A conjugated superantigen is a conjugate between an independent superantigen and a portion that looks for an immunomodulator. A conjugated superantigen exerts either SDCC and SADCC or both.

An immunomodulator (IM) is a compound capable of regulating the immune system. In the context of the invention, superantigens are treated separately and are included when the term immunomodulator is used. An immunomodulator often has the inherent ability to go to the target, similar to a corresponding lymphocyte receptor. Unless otherwise specified, an immunomodulator is in an unconjugated form.

A portion that searches the target (T) is a portion that is capable of joining a cell surface structure and / or a tissue structure.

A conjugate is composed of two or more portions Sag, IM, T, etc. that are linked to each other covalently. By soluble forms of active ingredients means forms of body-derived fluids that are soluble such as serum and plasma.

BACKGROUND OF THE INVENTION - THERAPEUTIC USE OF SUPERANTIGENS Unconjugated superantigens and wild type mutants have been suggested for therapy with a curative effect presumably achieved through an activation of the immune system, either locally in the cells expressing for class II associated with the disease to be treated or as a systemic activation (Kalland et al WO9104053; Terman et al WO9110680 and W09324136; Antonsson et al W09736932; and Newell et al 1991).

Due to the extreme toxicity of wild-type superantigens this approach, with respect to cancer treatment, should only be appropriate for a minor fraction of all cancers.

It has also been recommended to use the superantigens conjugated to the targeting portions of the target (Dohlsten et al WO9201470, Abrahmsen et al WO9601650, Antonsson et al W09736932 and Ochi et al 1993), all three publications are incorporated for reference).

In connection with studies of the prevention of down-regulation induced by a superantigen of T cell-mediated cytotoxic activity by IL-2 in vivo, it has been speculated that it should be beneficial to co-administer IL-2 with wild type superantigens. conjugates and with wild-type superantigens conjugated with antibodies (Belfrage Thesis Augusti / Septe ber 1996, Belfrage et al 1994, Belfrage et al 1995, Belfrage et al 1997a, Belfrage et al 1997b (wild type superantigens)), is also suggested. that the CD80 anchored to the cell membrane plays a role in the activation of the superantigen of T cells in the absence of MHC class II antigens (Lando et al 1993 and 1996).

Figure 4 in Lando et al 1996 shows an experiment in which the ability of a superantigen conjugated to an antibody alone or in combination with IL-2 to induce the proliferation of human T cells at rest was analyzed. In this 4-day experiment, the conjugated superantigen was presented to progenitor CHO-cells and transfected CHO-cells to express for a class II or C215 or a class II plus C215. The effect of IL-2 was negligible.

Kappler et al (W09314634) suggests that unconjugated and wild type SEB has mutated to lose its ability to bind to Vß or MHC Class II (in the context of vaccines and as an agent to neutralize the toxic effects of superantigens ). Abrahmsén et al (WO9601650) suggests that cancer therapy with conjugated superantigens that have a modified ability, or preferably decreased from a binding to Class II antigens. Antonsson et al (WE9736932) suggests a therapy with chimeric superantigens and superantigens with reduced serosensitivity (see also Abrahmsén et al). Mutations as described in Abrahmsén et al (WE9601650) and Antonsson et al (WE9736932) involve superantigens with reduced systemic toxicity, reduced immunogenicity and / or reduced serosensitivity for the mammal to be treated.

Therapy with the administration of nucleic acids encoding wild-type superantigens has been recommended by (Terman et al 1WO9110680; W09324136) and Dow et al WE9636366). Dow et al goes beyond the administration of nucleic acids encoding a cytokine or chemokine with a nucleic acid encoding a superantigen. Without faculty for experimental support, W09636366 also recommends structures in the form of a biscistronic gene structure in which one cistron contains the gene that codes for the superantigen and the other cistron contains the gene that codes for a cytokine or chemokine.

Without authorizing experimental support, Pouletty P (Sangstat EP 510949) speculates that conjugates between targeting portions of the target, such as IL-2 and wild-type superantigens may be useful for inactivating cells expressing for the IL-2 receptor.

BACKGROUND OF THE INVENTION-THERAPEUTIC USE OF IMMUNOMODULATORS E? COMBINATION WITH SPECIFIC ANTIBODIES FOR CELLS TO BE INACTIVATED.

It has been suggested in advance to conjugate antibodies with biological response modifiers, for example, a chemokine or a cytokine, similar to interleukin-2 (Fell et al EP 439095; Rosenblum et al EP 396387; Pancoo et al 1 96; and Becker et al. to 1996).

THE PROBLEM THAT THIS INVENTION IS WILLING TO RESOLVE.

The present invention is arranged to provide improvements in relation to the therapy of superantigens that involve the activation of the immune system in order to inactivate unwanted target cells in a mammal to be treated. In particular, the improvements relate to: 1) locally extending the activation period, for example, in a tumor, during a first cycle of treatment; 2) counteract the onset of hyposensitivity due to the tendency of activated T cells to escape in anergy; 3) facilitate the activation, in the tumor area, of the T cell independent of the MHC class II; and 4) expand the therapeutic window for cytolysis by means of superantigen activation. It has been discovered that these improvements can be carried out to their full or partial extent provided that the administration of a superantigen (SAG) is combined with the administration of an immunornodulator in soluble form, and at least one of the superantigens and the The immunomodulator is in the form of a conjugate with a portion that has properties that make it look for the cell to be inactivated.

THE FIRST PRINCIPAL ASPECT OF THE INVENTION; A METHOD OF INACTIVATING TARGET CELLS.

The first aspect of the invention covers both therapy and in vitro valuations and is a method to inactivate unwanted target cells in the presence of T cells, by putting the two cell types in contact with a superantigen ( SAG), in particular, a superantigen that activates T cells through a binding to a TCRVß, in the presence of an immunomodulator (IM) that is not a superantigen (Sag) In its broadest aspect, the method is characterized in that at least one of the superantigens and the immunomodulator is in the form of a conjugate with a portion (T) that has properties that make it look for the cell to be inactivated. In a subaspecto, the method is characterized in that a. the superantigen (SAG) and the immunomodulator are used as a triple conjugate comprising a superantigen (Sag), a portion that searches (T) for the target cells and an immunomodulator (IM) (conjugate-Sag, T, IM); b. the superantigen (SAG) is used as a double conjugate between a superantigen (Sag) and a portion that searches for (T) the target cells in -combination with a double conjugate between an immunomodulator (IM) and a portion that looks for (T ') ) to the target cells (conjugate-Sag, T + conjugate-IM T '); c. The superantigen (SAG) is used as a double conjugate between a superantigen (Sag) and a portion that searches (T) for the target cells and the immunomodulator (IM) is used independently, that is, it is not conjugated to a portion looking for the target cells (conjugate-Sag, T + IM); d. the superantigen (SAG) is used independently (Sag) and the immunomodulator is used in conjugated form, that is, a double conjugate between the immunomodulator (IM) and the superantigen (Sag) (Sag + conjugate-IM, T); and e. the superantigen (SAG) and the immunomodulator are used as a double conjugate between a sup'erantigen (Sag) and an immunomodulator (conjugate-IM, Sag); The superantigen and the immunomodulator can be made to look for the same cell type, for example, to identical structures / epitopes or to cross-react or to different cell types within the same tissue. Going to the target may be for normal cells or diseased cells associated with the same tissue. Any or both superantigene and immunomodulator can be made to one or several antibodies.

Diseases to be treated by the method of the invention.

The diseases to be treated are, in principle, the same as those previously suggested by superantigens. See for example, under the headlines of "Antecedents ..." mentioned previously. Illustrative examples are cancer, autoimmune diseases, parasitic infestations, viral infections and other diseases associated with cells which on their surface express MHC class II antigens and / or other structures that are specific for the respective diseases and which are they join the portion that looks for the target incorporated in the superantigen in accordance with the inventive concept (formula I). Also bacterial infections can be combated by the use of the invention.

Important forms of cancer in the context of the invention are: melanomas, carcinomas, hematopoietic neoplasms and fibrosarcomas that include specific forms such as squamous cell carcinoma, breast cancers, carcinomas of the head and neck, carcinomas of the thyroid, carcinomas of the soft tissues, bone carcinomas, testicular cancer, prostate cancer, ovarian cancers, bladder cancers, skin cancers, brain cancers, angiosarcomas, hemangiosarcomas, tumors of the mammary gland cells, primary liver cancers, lung cancers , cancer of the cervix, renal cell carcinomas, leukemias and lympholas. They include any type of benign or malignant tumors as well as cancers resistant to various drugs, metastatic cancers, various forms of chemically or virally induced cancers (Herpes, SV40, HIV, etc).

THE SECOND MAIN ASPECT OF THE INVENTION: INVENTIVE CONJUGATES OF SUPERANTIGENS.

This aspect of the invention comprises conjugates that comply with the formula (T) x (Sag) and (IM) z Formula IT is a portion that searches for the target, Sag corresponds to an independent superantigen and IM is an immunomodulator that is not a superantigen . T, Sag and IM are linked by organic linkers B which may be different or the same within one or the same substance or conjugated molecule. The conjugates according to formula I encompass the chemical conjugates as well as the recombinantly produced conjugates (fusion proteins). The x, y, z are integers that are typically selected from 0-10, such as 0-5 representing the number of T, Sag and IM portions respectively, in a given conjugate molecule, with the stipulation that y > 0 and also one or both of x or z > 0. Chemical conjugates are usually conjugated substances that contain a mixture of different conjugated molecules. Therefore, some substances of chemical conjugates x, y, z may also not be integers within the range 0-5.

In a first subassembly of the formula I, Sag and IM and T are presented in the conjugate (x, y, z> 0, conjugates-IM, T, Sag). X, y, z are typically integers of 1-3, preferably 1-2. The typical relationships between x, y, z are: x = y = z; x = y = 0.5z; x = 0.5 y = 0.5z; and x = 0.5y = z. In a second subaspecto of the con ugdos according to formula I, the portion that looks for the objective is absent (conjugated-Sag, IM, x = 0). The y, z are typically integers of 1-3. The preferred relationships between x, y are: x = y; x = 0.5y, 0.5x = y; x = l / 3y and l / 3x = y.

In both subaspects, integer or related numbers refer in the first instance to fusion proteins in which there is a T in each conjugate molecule. The formula I for the conjugates according to the second subaspecto is reduced to: (Sag) and (IM) z Formula II This type of conjugates are especially adapted to the treatment of diseases associated with diseases of cells that express for MHC class II antigens, in particular, cancers that express for class II such as cancers of the hematopoietic system, and certain diseases autoimmune, viral infections and parasitic infestations, but also for diseases associated with receptors anchored to the cell membrane for the immunomodulator, for example, T-cell lymphoma expressing, for example, the IL-2 receptor.

A. IM IMMUNOMODULATOR IM E FORMULA IM appears as an immunomodulator that is not an independent superantigen or conjugate. The immunomodulator can be a cytokine or chemokine. Illustrative cytokines are the granulocyte-stimulating factor of the macrophage colony (GM-CSF), the tumor necrosis factor a or β (TNFα or TNFβ), the macrophage colony stimulating factor (M-CSF), the stimulating factor of granulocytes (G-CSF), IL-1, IL-2, IL-4, IL-6, IL-7, IL-12, IL-15, IL-18 and IGF. Exemplary chemokines are C5a, IL-8, monolabeled chemoattractant lalfa protein (MlPlalfa) or l-monocyte chemotactic protein (MlPlβ), monocyte chemoattractant protein 1 (MCP-1), monocyte chemoattractant protein 2 (MCP-) 2), monocyte chemoattractant protein 3 (MCP-3), platelet activating factor (PAFR), N-formyl-methionyl-leucyl-phenylalanine (FMLPR), leukotriene B4 (LTB4R), gastrin-releasing peptide (GRP) ), RANTES, eotoxin, lymphotactin, IP10, 1-309, ENA78, GCP-2, NAP-2, MGSA / gro, DC-CKl, Flt3L (ectopic domain), fractalcin, PF-4, etc.

Another type of immunomodulators are those that are derived from ligand / receptor pairs anchored to the cell membrane and involved in the modulation of a triggered immune response, such as co-stimulation (eg, lymphocyte receptors anchored to the surface and corresponding ligands). attached to the cell). Illustrative examples are the members selected from the pairs CD40L / CD40, 4-BB1 / 4-BB1L, CD28 / P7, CDLA-4 / B7, etc. B7 includes variants such as CD80 and CD86 with preference for the former. The preferred forms are soluble, contain the extracellular part (ectopic domain) and are devoid of the intracellular or membrane anchored parts.

Particularly preferred immunomodulators are capable of potentiating the effects of superantigens in vivo, for example, by counteracting the escape in anergy of T cells activated by superantigen. Ligands / receptors bound to typically appropriate cells are CD28 / B7 and include analogs and fragments as defined above. The typical cytokines of this group are IL-2, being the main effector of the 3 'direction of the signaling of CD28 / B7, and of the cytokines similar to IL-2; IL-7 and IL-15. Among the ligand / receptor pairs associated with the surface of T cells, it is preferred that the non-T cell member to be activated is incorporated into a conjugate according to the invention. For CD40L / CD40, 4-BB1 / 4-BB1L and CD28 / B7 this means soluble forms of CD40, 4BB1L and B7 with preferences as defined above. The experimental part of this text illustrates the variants of immunomodulators that at the priority date were found to be optimal within the invention.

Immunomodulators should preferably be of the species that of the individual being treated. Natural immunomodulators, such as cytokines and chemokines, generally show high systemic toxicity and a relatively short half-life in mammals. The literature is extensive on how to modify the immunomodulators for an increased stability relative to the oxidation, a longer half-life in vivo, a lower toxicity, an enhanced redoubling when it is produced by genetic recombination techniques, etc. For example, US 5, 229, 109 (Grimn et al) describe low toxicity analogs for IL-2 that have a reduced affinity for the high affinity IL-2 receptor (IL-2R) because they are deficient in binding with the p55 subunit of the receptor. Analogs are prepared primarily by mutating the codon for an amino acid at position 33-46 in IL-2 (e.g., Arg38Ala and Fe42Lis or Fe42Ala). The Asp20Lis mutant has a 100-500 fold reduced affinity for the β-chain / p75 of the IL-2 receptor without affecting binding to p55 (Collins et al 1988). Other mutations, for example, Asp20Ser are less severe (Berndt et al 1994). Studies in murine IL-2 indicate that Asp84 and Asn88 of human IL-2 are also involved in the binding of p75 (Zurawski et al 1993). This is supported by manipulating the binding between human IL-2 and its receptor (Baborough et al 1994). The expected reduction in the affinity of these mutations is Asp20 >Asn88 > Asp84.

Combining the mutations in Arg38 and Fe42 with mutations at position 88 and 20 would result in even a lower affinity for IL-2R. Another powerful mutation and to date of preferred priority of IL-2, is Tr51Pro which produces an analogue of IL-2 with a lower proportion of cellular internalization and a prolonged duration of its immunomodulatory effect (Chang et al 1996). The numbering of amino acid positions in accordance with Taniguchi et al 1983.

The publications by Gri et al; Collins et al 1988; Berndt et al 1994; Zurawski et al 1993; Bamborough et al 1994; Chang et al 1996; and Taniguchi et al 1993 are incorporated for your reference.

The use of cytokine analogues and chemokine with a reduced affinity for their normal receptors in conjugates as described above will reinforce their ability to go to the target to the preselected target cell. It is conceivable that the cytokines and chemokines have mutated to show a reduced proportion of cellular internalization at the time of binding to their respective cellular receptor and incorporated into a conjugate which, according to formula I, will result in a prolonged superantigen activity compared to the corresponding conjugates that they have the natural form of the immunomodulator.

The term immunomodulatory (IM) in this manner encompasses any modified form, e.g., any form that has mutated and that is capable of agonizing or antagonizing the effects of the corresponding natural form of the immunomodulator.

B. THE SAG PART OF SUPERANTIGEN IN FORMULA I.

The SAG in formula I represents a superantigen as defined for the independent superantigens and under the heading of "background ..." mentioned above, that is, to wild-type superantigens possibly modified, for example, by a mutation, to. to have a decreased ability to bind a MHC class II antigen compared to the corresponding wild-type superantigen (see, for example, in Abrahmsen et al (WO9601650)).; b. to have a decreased serorsensitivity in human serum compared to the corresponding wild-type superantigen (see for example, Abrahmsén et al (WO9601650) and Antonsson et al W09736932 and Antonsson et al 1997); c. to have a decreased immunogenicity in humans compared to the corresponding wild-type superantigen (see for example, Antonsson et al W09736932 and Antonsson et al 1997); d. to be a chimera between two or more wild type analog superantigens wherein a region in a first wild-type superantigen has been replaced with the corresponding region in a second analog superantigen. The region in question may be a region that determines binding to TCRVβ, for example, as defined for the SEE / SEA and SEA / SEE chimeras (see for example, Antonsson et al W09736932, Antonsson et al 1997 and Lamphaer et al 1996 ).

Other modifications / mutations that may be found to be appropriate are also included, for example, to prevent glycosylation when it occurs in eukaryotic cells.

Typical mutations to priority of superantigens similar to SEA / SEE were (numbering as used by Antonsson et al 1997 and Antonsson et al W09736932): a. a decreased MHC class II binding: Asp227Ala (= SEAm9), Fen47Ala and / or Asp70Arg. He Fen47Ala / Asp22Ala = SEAm23 mutant. b and d. The chimeras between SEA and SEE, aim to reduce serosensitivity in humans, while retaining the SADCC capacity of the corresponding conjugated superantigens: SEE with the following substitutions: Gly20Arg, Tr21Asn, Gly24Ser, Lis27Arg. The mutants used in the experimental part are SEA (Asp227Ala) = SEAm9, SEA (Fen47Ala / Asp227Ala) = SEAm23 SEA (Fen47Ala / Asnl02Q / Asnl49Asp / Tr218Val / Asp227Ala) = SEAm57. In the priority cataloging the preferred superantigens were selected from: 1. superantigens (Sag) exhibiting two MHC class II binding sites (eg, staphylococcal enterotoxins A and E), 2. superantigens (Sag) than in their form that has not mutated required a Zn-ion for its optimal binding to MHC class II (eg, SEA, SEE and SEH), 3. staphylococcal enterotoxins.

A Sag molecule that is incorporated into a conjugate according to the invention does not need to be a functional superantigen, the main issue being that the final conjugate is so when exercising either or both of SADCC and SDCC as outlined above.

C. PORTION YOU ARE LOOKING FOR IN FORMULA I.

T can, in principle, be any structure that is capable of binding to a cell surface structure, preferably a specific structure of a disease. The structure against which T is directed is usually different (a) from the polymorphic epitope of the chain TCR to which binds Sag (b) of the MHC class II epitopes to which Sag binds. The targeting portion can be selected from interleukins (e.g., interleukin-2), hormones, antibodies that include antigen-binding fragments of antibodies, growth factors, etc. See for example, Woodworth 1993 (which is incorporated here for your reference). The portion that looks for the target can thus be a protein containing 1, 2, 3, or 4 polypeptide chains.

On the priority date, it was preferred that T be an antibody (full length antibody, FAB, f (AB) 2, Fv, ScFv (single chain antibody), multiple single chain (ScFv) n antibodies and any other antigen-binding antibody fragment), including any truncated, functionally active form of the aforementioned forms of antibodies. Other variants are monospecific and bispecific. The antibody can in principle be directed towards any specific / associated cell surface structure of a disease, eg, structures provided previously linked to any form of cancer, with particular emphasis for the active fragments of antibodies (such as Fab). Typically, the antibody can be directed towards the colon and / or specific epitope of the pancreas, for example, the so-called epitope C242 (Lindholm et al WO9301303), a specific epitope for lung cancer, for example, the epitope for the 5T4 antibody (Stern et al WO8907947), a specific epitope of lymphoma, for example, in CD19, a specific epitope of melanoma, eg, HMW-MAA, etc. The term "antibody" comprises monoclonal as well as polyclonal variants, preferably for monoclonal preparations.

In case the target portion is a Fab fragment the cystine residues, this normally binds the heavy and light Fab chains, preferably it has been replaced by an amino acid which does not allow the formation of disulfide, for example, serine. See also Antonsson et al W09736932.

What has been said previously also includes that T can be directed towards unique structures in more or less healthy cells that regulate or control the development of a disease.

D. THE LIGATOR B.

Binder B may be selected as previously described (Dohlsten et al WO9201470; Abrahmsen et al WO9601650; and Antonsson et al W09736932), ie B must preferably be hydrophilic and exhibit one or more structures selected from amide, thioether, disulfide , etc. The most prominent linkers are those obtained by genetic recombination techniques, ie, that the conjugation is carried out at the genomic level resulting in oligopeptide linkers. Typically oligopeptide linkers contain from 1-30, such as 1-20 amino acid residues which are preferably selected so that the linker is hydrophilic in its entirety.

Preferably, the residues of the linkers are selected from hydrophilic amino acid residues, such as Gly, Ser, Gly, Glu, Pro, His, and Arg. Typical oligopeptide linkers comprise the tripeptide GlyGlyPro or the so-called Q linker (Wootton et all989 incorporated in the reference) that possibly is modified with Gly-pro at its amino terminal end.

E. LINK POINTS FOR T, SAG AND IM.

The chemical conjugates will typically contain a mixture of conjugated molecules that differ in the binding positions. The conjugated substance contains hetero-conjugates as well as ho-conjugates.

For recombinant conjugates (fusion proteins) the conjugate substance obtained will be uniform with respect to the position of the enalce. For each individual subunit (T, Sag, IM) the amino terminal is fused to the carboxyl terminal of another subunit or vice versa, preferably by means of an inserted oligopeptide bridge. The combinations in case the conjugate contains one of each T, IM, Sag will be T-IM-Sag, IM-T-Sag, Sag-IM-T, T-Sag-IM, IM-Sag-T (the occurrence of a link structure for B is not shown). In case one or more of the subunits contains two or more polypeptide chains, the number of possibilities increases: for T which is a pair fragment of antibody, the possibilities will be (B linkers of oligopeptides are not shown): 1. Sag-Fab (light chain) -IM 2. Fab (cadenaliviana) 'Fab (heavy chain) Sag-Fab (heavy chain) -IM 3. Sag-Fab (light chain) 4. Fab (light chain) -IM Fab (heavy chain) -IM Sag-Fab (heavy chain) 4. Sag-Fab (light chain) 6. IM-Fab (light chain) IM-Sag (heavy chain) Sag-Fab (heavy chain) 7. Fab (light chain) -Sag 8. Fab (light chain) -IM Fab (heavy chain) -IM Fab (heavy chain) -Sag In other variants, the immunomodulator and superantigen can be fused in sequence to any extremity in any of the antibody chains.

At the priority date, recombinant conjugates are preferred, most preferably for even fragments as target-seeking portions and bonds of the amino terminal of the superantigen independent to the first constant domain of the heavy (C, 1) or light chain of antibodies and the remaining carboxyl terminal immunomodulator (valid for formulas I-IV).

For optimal function and production, the fusion protein is recombinantly expressed as a two-chain product in which the superantigen is fused in C-terminal form to the C-domain, 1 of the Fab fragment of the antibody by means of a 3-11 residue hydrophilic amino acid linker. This linker may have the sequence Gly-Gly-Pro or Pro-Ala-Ser-Gly-Gly-Gly-Gly-Ala-Gly-Gly-Pro (identification of sequence No: 19) or 4-9 residues based on Id SEQ No: 19, ID SEQ No: 19 being preferred. The immunomodulatory portion is fused C-terminally to the light chain by means of a hydrophilic linker and neutral or positively charged 10-20 residues (Q linker) preferably, the linker Q can have the following sequences Gly-Pro-Arg-Gln-Ala-Asn-Glu-Leu-Pro-Gly-Ala-Pro-Ser-Glu-Glu-Glu-Arg (SEQ ID No: 23), Gly -Pro-Arg-Gln-Ser-Asn-Glu-Tr-Pro-Gly-Ser-Pro-Ser-Gln-Glu-Glu-Arg (SEQ ID No: 20), Gly-Pro-Arg-Gln-Ala Lis-Tr-Leu-Pro-Gly-Ala-Pro-Ser-Gln-Tr-Tr-Arg (SEQ ID No: 21) or Gly-Pro-Tr-Glu-Ala-Asp-Glu-Leu-Pro-Gly -Ala-Pro-Ser-Glu-Glu-Glu-Tr (SEQ ID No: 22), with SEQ ID No: 20 and 21 being most preferred (see Example 2 for more details).

It is believed that at this stage analogous combinations at the amino terminus or the combination of linkages at the amino and carboxyl terminals of the VH and VL domain resulted in active but less efficient conjugates.

F. The active entities that do not comply with the formula but that are used in accordance with the combinations a-e mentioned above in the inventive method.

Independent superantigens (Sag): See under the headlines "definition". Typical Sags are determined under the headings of "Background" and "B. The superantigen part of Sag in formula I".

Non-conjugated immunomodulators: See under the heading of "Definition". In principle, the same immunomodulators that are determined under the heading "A. Immunomodulator IM in formula I" can be used. Emphasis is placed on the preference for cytokines and chemokines for IL-2 and for cytokines similar to IL-2 Objective immunomodulators (with ugados-T, IM): These conjugates comply with the general formula: (T ') x (IM') z Formula III in which T 'and IM' are linked by an organic linker B '. The t', IM 'and B' are selected from the same groups of structures / compounds of T, IM and B. The x, z are defined in the same manner as in formula I (y = 0), preferably for one or two IM 'by T' and conjugated molecule. The points of union between T 'and IM' are as defined for formula I. See also under the headline "E. Points of union ..." where formulas 1-8 and comments to them also apply to conjugates T, IM except for Sag that is omitted. The conjugates of formula III can be manufactured according to known techniques, that is, conventional techniques of chemical binding or genetic recombination (fusion proteins) with preference for the latter (Fell et al EP439095; Rosenblu et al EP396387). The conjugates-IM, T comprise a portion -IM 'which has been modified, for example, which has been mutated, to have a reduced affinity and / or reduced internalization rate as defined above.

Target superantigens (conjugated-T, Sag): These conjugates comply with the general formula: (T ") x (Sag") and Formula IV In which T '' and Sag '' are linked by the organic linkers B ''. The T '', Sag '' and B '', are selected from the same groups of structures / compounds as T, IM and B. The x, and are defined in the same way as in the formula I (z = 0 ), preferably for one or two Sag '' by T '' and a conjugated molecule. The junction points between T '' and Sag '' are defined for formula I. See also under the heading "E. Join points ..." in which formulas 1-8 and the comments of them apply also for conjugates T, Sag except that IM is omitted from the formula. The conjugates of formula II can be manufactured according to known techniques, i.e. techniques of genetic recombination or conventional chemical bonds (fusion proteins) with reference to the foregoing. See, for example, Dohlsten et al WO9201470; Abrahmsén et al WO9601650, Antonsson et al WO 9736932.

Third main aspect of the invention: conjugates containing a modified immunomodulator.

These novel conjugates comply with the formula: (T "') x (Sag'") and (IM "') z Formula V In which T '' 'and Sag' '' are selected from the same compounds of T and Sag of formula I. IM '' 'is an immunomodulator that has been modified, for example, by a mutation, so that exhibits a decreased affinity for the receptor anchored to the cell membrane and / or for a decreased rate of internalization by a ligand to its receptor (compared to the corresponding natural forms). The IM "is preferably a cytokine or a chemokine. See below under the heading "A. IM immunomodulator in formula I". An important immunomodulator for this aspect of the invention is modified IL-2. The x, y, z are defined in the same way as in formula I.

In a first subassembly of the conjugates according to the formula V T '' ', Sag' '' and IM '' 'are always present (all x, y, z> 0), that is, conjugates T, Sag, IM. Typically, x, y, z are integers 1-3, preferably 1-2. The typical relationships between x, y, z are: x = y = z; x = y = 0.5z; x = 0.5y = 0.5z; and x = 0.5y = z.

In a second subaspect of the conjugates according to formula V, the portion of the superantigen that is absent (y = 0), that is, conjugates T, IM. The x, z are typically integers 1-3. The preferred relationships between x, z are: x = z; x = 0.5z; 0.5x = z; x = l / 3z; and l / 3x = z.

In both subaspects of the conjugates according to formula V, the ranges determined for the integers and their interrelationships refer primarily to the fusion proteins in which the portion that looks for the target can be a protein that contains 1, 2, 3 or 4 polypeptide chains and in which there is a T in each conjugated molecule. The binding sites in the fusion proteins of formula V are the same as for the corresponding fusion proteins that comply with formula I. See also under the heading "F. Binding points ..." in which the formulas 1-8 and their comments also apply to conjugates T, IM except that, for the second subaspect, Sag is omitted.

Fabrication of the conjugates as defined above.

In principle, the manufacture of the conjugates is. It can be carried out according to two main routes: 1. Chemical union of the individual subunits T, Sag and IM together. Each individual subunit or combination thereof can be produced by genetic recombination techniques. 2. Genetic recombination techniques that directly provide a conjugate that is defined in any of the previously determined formulas.

The precise methods are recognized for the average skilled worker and comprise many variants. The chemical linkage typically utilizes functional groups (eg, primary amino groups, carboxyl groups, mercapto groups, carbohydrate groups) that are typically present naturally in various positions of superantigens, protein immunomodulators and in protein portions that seek objective. The methods are well known in the art.

The major host cell for a large-scale genetic recombination production of the inventive conjugates between a superantigen and an immunomodulator (fused forms as well as unconjugated forms) is E. coli. This host provides in principle two routes: intracellular production and secretion. The latter variant is preferred because it offers a purification of the correctly folded proteins of the periplasm and the culture medium. The foregoing does not exclude the possibility of also producing active conjugates in other host cells, for example, eukaryotic cells, such as yeast or mammalian cells. Preliminary results indicate that up to now, eukaryotic cells such as mammalian cells may be preferred to incorporate immunomodulators that interact with CD28, eg, B7 and its analogues.

The fourth aspect of the invention: gene structures coding for the new inventive conjugates A fourth aspect of this invention is a recombinant nucleic acid molecule, preferably DNA, such as cDNA, or RNA, which codes for a superantigen and an immunomodulator as defined above, preferably for IL-2, IL-7, IL -12, IL-18, RANTES, ectopic domain of CD80, ectopic domain of CD86, 4-BB1L and Flt3L and their analogs as defined above. In this aspect of the invention, the nucleic acid must typically contain control regulatory sequences such as translation control sequences, origin of replication, signal secretory sequences for either or both immunomodulators and the superantigen, etc. ., which are compatible with the host cell in which the immunomodulator and the superantigen are expressed. The region between the parts of sequences encoding the superantigen and the immunomodulator may comprise a sequence encoding ribosome entry sites such as in biscistronic gene structures. The later will allow a separate expression. In case of combinations of multiple chain conjugates containing for each chain of polypeptides that is comprised within the conjugate IM, Sag, a portion of conjugates seeking the target, the appropriate signal sequences, the biscistronic structures will facilitate the dubbing and assembly of active conjugates with an IM bound to one of their chains. This is important for cases where the target-seeking portion is a double-chain antibody molecule and at least one of the superantigens (Sag) and the immunomodulator are fused to a terminal end of the antibody chain. See the above PHARMACEUTICAL COMPOSITIONS, DOSAGE AND ADMINISTRATIVE ROUTES.

A fourth aspect of the present invention is a pharmaceutical composition containing the inventive combination of the superantigen (Sag), the target seeking portion (T) and the immunomodulator (IM). The characteristic feature of this aspect of the invention is that at least one superantigen and one immunomodulator is in the form that allows one to go to the target or go to the cells to be inactivated as described above.

The compositions contemplated are known in the art, except that they now contain the combination of the inventive conjugate. In a particular composition the composition should comprise: a. a triple conjugate comprising a superantigen (Sag), a portion seeking (T) the target cells and an immunomodulator (IM) (T, IM, Sag conjugate); b. two double conjugates - one between an antibody that searches (T) and a superantigen and the other between an antibody that searches (T ') and an immunomodulator (ie, conjugate-T, Sag + conjugate-T ', IM)'. The T and T 'may be different or equal in regard to the epitope specificity. c. a double conjugate between a superantigen (Sag) and a portion that looks for the target (T), and an immunomodulator (IM) independently (conjugate-T, Sag + IM); d. a double conjugate between an immunomodulator (IM) and a target-seeking portion (T) and an independent superantigen (Sag) (conjugate-T, IM + Sag); or a superantigen (Sag) and an immunomodulator (IM) in the form of a double conjugate (conjugate-Sag, IM); See more back in the context of inventive methods. In case the compositions contain two active components (b-d mentioned above) the composition can allow the maintenance of the components apart until the occasion of their administration.

The compositions can be as a lyophilized particulate material, a sterile solution or aseptically produced, a tablet, a vial, etc. Vehicles such as water (preferably being buffered to a value with a physiologically acceptable pH by eg PBS) another inert solid or liquid material may be present. In general terms the compositions are prepared by the conjugate, possibly in combination with a non-conjugated active component, which can be mixed with, dissolved in, bound to, or otherwise combined with one or more water soluble or non-soluble carriers. water, aqueous or non-aqueous, and if necessary together with appropriate adjuvant additives. It is imperative that the vehicles and conditions do not adversely affect the activity of the active compounds as defined in the above-mentioned a-e points.

Normally the superantigens (Sag) that are used in the invention will be sold and administered in doses prepared in advance, each containing an effective amount of Sag which, based on the result that is now presented, is thought to be within the range of lpg. -50mg. The exact dose will have case variants in case depending on the weight of the patient and their age and their pre-titration of specific antibodies for the Sag to be used, route of administration, type of the disease, target seeking portion, superantigen, union (-B-), immunomodulator, etc.

An important factor to consider in the determination of a dose for a combination to be used in the inventive method is that superantigens and immunomodulators exercise optimal dose ranges. A very low dose will result in a suboptimal effect or will have no effect and a very high dose will give unacceptable side effects such as toxicity which can be lethal. In this way, the broad range determined previously can be emphasized in an attempt to encompass all possible ranges of all the variants of the inventive method. In this way, each specific combination according to the inventive method has a subrange of doses within the range of 0. lpg to 50 mg. This does not exclude that future evolutions and results can be carried to dose levels outside this range.

The routes of administration will be those that are commonly contemplated within the field, i.e., an effective amount or therapeutically active amount that annihilates a combination of an antigen-immunomodulator that according to the invention contacts the target cells. For the indications specified above, this means that primarily a parenteral administration, such as an injection or infusion (subcutaneously, or intravenously, intrarterially, intramuscularly, intraperitoneally) in a mammal, just like a human being. The combination of the conjugate of the invention can be administered locally or systemically.

By the term "effective annihilating amount of the target" it is contemplated that the amount is effective to activate and direct the T cells to destroy the target cells.

Preferred administration routes at the priority date are the same as those contemplated for conjugates of superantigens according to Dohlsten et al WO9201470; Abrah sen et al WO9601650 and Antonsson et al PCT / 97/00537. This means during 1-5 hours of intravenous infusion (preferably for 4 hours) per day in combination with a fever reducing agent (paracetamol) the administration will be repeated a few days, for example, for 5-8 days with a consideration being taken for the risk of an antibody stimulation directed towards the conjugate. Optimally, that there are several cycles of therapy with each cycle containing a treatment during a space of one or more days followed by a rest period for one or more days, for example, cycles with treatment with rest for two days, respectively.

The inventive compositions can be administered either as a main therapy or preferably as adjuvant therapy in connection with surgery or with other drugs.

EXPERIMENTAL PART Registration to the figures Figure 1. It is a FACS analysis of the CHO-CD28 cells stained with CD80-C215Fab, CD80-C215-Fab-SEAm57 or C215Fab-SEA followed by an incubation with an anti-mouse kappa mAb chain labeled with PE. The staining was done at 4 ° C without washing. Ordered: Medium channel.

Figure 2. It is FACS analysis of Colo205 cells stained with CD80-C215Fab, CD-80C215Fab-SEAm57 or C215Fab-SEA followed by incubation with a kappa mAb chain of PE-labeled anti-mouse. The staining was done at 4 ° C with three washes between each staining step. Ordered: Medium channel. Abcisa: Effector (E) for the proportion of the target cell (T).

Figure 3. It is a proliferation. The T cells are incubated with Colo205 cells. and 4uM C242Fab-SEA and variable amounts of CD80-C215Fab for 4 days after the incorporated -l-thymidine was counted. The amount that was obtained without any CD80-C215Fab was 20039 cpm +/- 1750. Figure 4. Shows the production of IL-2. T cells were incubated with Colo205 and 4uM C242Fab-SEA cells and varying amounts of CD80-C215Fab for 4 days after the supernatant was depleted and the amount of IL-2 was determined. The amount of IL-2 that was obtained without any CD80-C215Dfab was 2849 pg / ml. Figure 5. Shows the proliferation. T cells were incubated with Colo205 cells in varying amounts of CD80C215Fab-SEAm57 'or C215Fab-SEAm23 for 4 days after the incorporated thymidine-JH was counted.

Figure 6 is the production of IL-2. T cells were incubated with Colo205 cells in varying amounts of CD80C215Fab-SEAm57 or C215Fab-SEAm23 for 4 days after the supernatants were depleted and the content of IL-2 was determined.

Figure 7. Proliferative capacity of T cells of human blood that were incubated for days with the indicated proteins and with the irradiated and transfected CHO cells (for the presentation of the SEA). Ordered: incorporation of 3H-thymidine (cpm).

Figure 8. It is the simultaneous administration of SEA and IL-2 that seeks to lead to an intensified activation of T cells in vivo; cytotoxicity (expressed as a percentage) against RAJI cells in MHC class II SEA-coated mouse spicules treated with one or three injections of C215FabSEA (FS), C215Fab-Q-hIL-2 (Fl) , a combination of the two (FS + Fl) or C215FabSEA-Q-hIL2 (FSI). Effector: the proportion of the target cells was 30: 1, and the cytotoxicity was quantified to a standard titration with a 51 Cr release for 4 hours.

Figure 9. It is the therapy of 5-day B16-C215 tumors in C57B1 / 6 mice with three injections (days 5, 6 and 7 after tumor inoculation) of C215FabSEA (FS), C215Fab-Q-hIL2 ( Fl), C215FabSEA + C215Fab-Q-hIL-2 (FS + Fl) or C215Fab-Q-hIL-2 (FSI). Equimolar amounts of. Fab-Q-hIL2.

Abcisa: amount of protein injected. Ordered: reduction of the tumor expressed as a percentage.

Figure 10. It is the increased infiltration of the CD25 T cell tumor followed by a treatment with C215FabSEA (FS), C215Fab-Q-hIL2 (Fl or C215FabSEA-Q-IL2). Cells positive for CD-25 that infiltrate the lung of a mouse carrying established B16-GA733 lung tumors were estimated by immunohistochemistry. Ordered: percentage of the stained area. Abcisa: 1 = TVS; 2 = first injection; 3 = second injection; 4 = third injection; 5 = fourth injection.

Figure 11. Are the sustained Interferon levels? after four injections, once a day (37 mg / injection), of C215FabSEA-Q-hIL2 (37 g /). The blood was collected four hours after the last injection and the content of Inferieron was determined? (in the ordinate) by means of a quantification of ELISA using Inferieron? Murine recombinant (Pharmingen) as a standard. A similar experiment was carried out with equimolar amounts (30mg / injection) of C215FabSEA (FS).

Figure 12. Cytotoxicity (expressed as a percentage of the ordinate) against MHC class II Raji cells coated with SEA from mouse spikelets treated with PBS or, 4 or 6 injections of C215FabSEAD227A- Q-hIL2 (= FSm9-IL2). The cytotoxicity was quantified in a standard 51 Cr release for four hours. PBS is used as a negative control.

Figure 13 is the 3-day B16-C215 tumor therapy in C57 Bl / 6 mice followed by a treatment of 8 injections of C215FabSEAc -A-Q-hIL2 (= FSm9-IL2) or C215FabSEAD227 (= FSm9). The treatment was given daily for 8 consecutive days. On day 21 the animals were sacrificed, and lung metastasis was counted. Ordered: reduction of the tumor that is expressed as a percentage.

Figure 14. Tumor therapy is B16-C215 for three days in transgenic Vb3 TCR mice C215FabSEAD227A-Q-hIL2 (= FSm9-IL2 (F42A)), C215FabSEAD227A-Q-hIL2 (= FSm9-IL2) or C215FabSEAD227A-Q -hIL2 (= FSm9-IL2). The treatment was given daily for 8 consecutive days. On day 21 the animals were sacrificed, and lung metastasis was quantified. Ordered: reduction of the tumor expressed as a percentage.

Figure 15. Three-day B16-C215 tumor therapy in transgenic Vb3 TCR mice followed by a treatment of 8 injections of C215FabSEAD227A-Q-hIL2F42A (F42A), C215FabSEAD; _. 7A-Q-hIL2 F42A F42K or C215FabSEAD227A -Q-hIL2Fj2A / D2üs (F42A / D20S). The treatment was given daily for 8 consecutive days. On day 21 the animals were sacrificed and the lung metastasis was quantified. Ordered: reduction of the tumor expressed as a percentage.

EXAMPLE 1. BIOLOGICAL ACTIVITY OF THE FUSION PROTEINS CD80-C215FAB FOR YOUR USE IN THE COESTIMULATION OF TUMOR THERAPY WITH FAB THAT GOES TO SAG.

Brief Description of the Invention: The CD80-C215Fab and CD80-C215Fab-SEAm57 fusion proteins have been constructed to contain the extracellular domain of human CD80. The fusion proteins were produced in mammalian cells, were produced to bind to CD28 using CHO cellular transfectants and to the C215 antigen using Colo205 cells. Both fusion proteins bound to CD28 and to the C215 antigen, the subsequent binding being 100 times lower compared to C215Fab-SEA. Since CD80 is bound to the N-terminal portion of the L-chain it is possible that the CD80 portion interferes with the binding of C215Fab. Both fusion proteins co-stimulate activated SAG T cells in humans and show that soluble CD80 retains its biological function.

MATERIAL AND METHODS Recombinant DNA techniques and enzymes: Plasmid DNA preparations and other separations were carried out essentially according to Sambrook et al (Sambrook et al 1989). E. coli HB101 was used (Boyer et al 1969) as the host strain. The restriction endonucleases and Klenow fragment of DNA polymerase I were obtained from Boehrlnger Mannheim or New England Biolabs and used according to the suppliers' recommendations. The polymerase - Taq was obtained from Perkin Elmer. The cDNA was made from total RNA using the GeneAmp RNA PCR kit (Perkin Elmer). The oligonucleotides were synthesized in a Gene Assembler (Pharmacia Biotech AB) or an ABI392 DNA / RNA synthesizer (Applied Biosystems), and purified by reversible phase chromatography in the FPLC system (Pharmacia Biotech). The sequencing was carried out according to the chain termination principle and dideoxyl (Sanger et al. 1977) using a set of sequencing tools of the completion cycle of Taq dideoxil from Applied Biosystems. The products were separated and detected in an ABl 373 DNA sequencer. A (Applied Biosystems). Bacteria harboring different plasmids were selected from plates containing 2 2 X Yt and 15 g of base agar per liter, supplemented with 70mg / L kanamycin or 100 mg / L ampicillin. The liquid broth is 2 X YT (per liter: 10 g of yeast extract (Difco), 16 g of Tryptone (Difco) and 5 g of NaCl) Table I LAKQ5 ATA TAA GCT TCC ACC ATG GGC CAC ACA CGG AG (SEQ ID NO: 1) LAKQ7 ACG CAG ATC TTT AGT TAT CAG GAA AAT GCT CT GC (ID SEQ NO: 2) LAKQ30 TCA AAG CTT CTC GAC CGC GCT GTT ATC AGG A ATG CTC (SEQ ID NO: 3) LAKQ37 CGC GCG TCA GGC TAA CGA ACT GCC AGG CGC C GTC ACA GAG ACG A (SEQ ID NO: 4) LAKQ38 AGT TTC GTC TCA CGC GCG TTC TTC CTG TGA C GGC GCC TGG CAG TTC GTT AGC CTG ACG (ID S NO: 5) KAKQ88 TGG TAC ACC ACA GAA GAC AGC TTG TG TAT G TG (SEQ ID NO: 6) LAKQ89 CAT ACA TAC AAG CTG TCT TCT GTG GTG TAC (SEQ ID NO: 7) LAKQ90 CGA ATA AGA AAG ACG TCA CTG TTC AGG AGT T (SEQ ID NO: 8) LAKQ91 CCA ACT CCT GAA CAG TGA CGT CTT TCT TAT T (SEQ ID NO: 9) LAKQ92 GAG ATA ATA ATA TTA TTA ACT CAG AAA ACA T (SEQ ID NO: 10) LAKQ93 CAT GTT TTC TGA GTT AAT AAC TTT ATT ATC T (SEQ ID NO: 11) LAKQ108 CGC GCA TCC GCG CGG CAC CAG GCC GCT GTT A CGG AAA ATG CTC TTG C (SEQ ID NO: 12) LAKQ117 CCG GAT AAC AGC GCG CGT CAG CTA ACG AAC T AGG CGC CCC GTC ACA GGA AGA CGC CCG CAG G CAÁ CTG CA (SEQ ID NO: 13) LAKQ118 GTT GGA CCT GCG GGC GTT CTT CCT GTG ACG G CGC CTG GCA GTT CGT TAG CCT GAC GCG CGC T TAT (SEQ ID NO: 14) Plasmid constructions Genes encoding a Fab fragment containing the variable domains of the murine C215 antibody were assembled as previously described (Dohlsten et al 1994). The cloning and mutagenesis of staphylococcal enterotoxin A (SEA) by means of genes producing the substitutes F47A and D227A have been previously described (Abrahmsen et al 1995). Three other mutations were introduced into the SEA gene using the primary primers LAKQ88-93 (all oligonucleotides used are compiled in Table I) to obtain a mutant SEA 57. LAKQ5 and LAKQ7 were used in RT-PCR to introduce a box of Kozak with 5 'direction, and to clone the gene coding for the identification peptide and the extracellular part in human CD80 from a total RNA of a human spleen. The nucleotide sequence was found to correspond to the CDO gene bank sequence (entry number M27533). Two variants of the CD80 gene were made, different cloning sites at the 3 'end: in the separated PCR the primary primers LAKQ30 and LAKQ108 were used to obtain a BssHII or Meol site, respectively. A fusion of genes coding for a CD80 which was fused before the kappa chain by means of a -Q linker (Wooton et al 1989), was constructed by inserting a DNA linker LAKQ37 / 38 BssHII-Esp3I, between the relevant CD80 gene and the Dsal site preceding the kappa gene. Plasmid pKGE987 was obtained by inserting the gene fusion coding for CD80- (Q-linker) -kappa, preceded by the CD80 indication peptide in a vector, which in addition to a CMV promoter and a poly-A end which contains a neomycin gene to be used for the selection of transformants. The last version of the CD80 gene was used to construct a gene fusion where it precedes the fusion of 57 Fd-SEA mutant genes: a DNA fragment (LAKQ117 / 118) encoding a Q-linker was inserted between the Mrol site in the CD80 gene and in a PstI site in codon 4 and 5 in the C215 VH gene. This series of genes was inserted into a second CMV promoter vector to produce the plasmid pMB189, thus coding for the CD80 signal peptide and the extracellular portion followed by a 57 Fd mutant and SEA, which are connected by the three GGP separator. waste. In the plasmid pKGE961, the Fd gene has been inserted following an indicator sequence (which is derived from another murine VH gene). Plasmid pMB156 codes for the natural kappa chain, preceded by its natural signal peptide, and contains the neomycin gene.

PRODUCTION 293 Hamster embryonic kidney cells were transfected with either pKGE961 and pKGE987 or with pMB156 and pMB189, to obtain cell lines that produce CD80-C215Fab, and CD80-C215Fab-SEAm57, respectively. To obtain stable cell lines, the selection medium was DMEM without phenol red (Pharmacia no MS 0127) supplemented with L-glutamine (GIBCO BRL no 25030-24), 10% serum of caliph of cattle and 1 mg / ml of Geneticin. The production medium was DMEM without phenol red supplemented with L-glutamine and 0.1% HSA (Pharmacia% Upjohn AB, Sweden). The fusion proteins were purified from the filtrate culture medium (Sartobran 0.65-0.4 um) by affinity chromatography on the Sepharose FF on the G protein (Pharmacia Biotech AB), followed by an anti-CD80 compatibility purification (anticorpo Anti-human immobilized CD80: Camfolio L307.4) or ion exchange chromatography on Sepharose FF (Pharmacia Biotech).

Reagents The RPMII 1640 medium (Gibco, Middlesex, England) supplemented with 2 mM L-glutamine (Gibco, Middlesex, UK), 0.01 M 'HEPES (Biological Industries, Israel), 1 mM NAHC03 (Biochrom KG, Berlin, Germany), 0.1 mg / ml gentamicin sulfate (Biological Industries, Kibbutz Beit Haemek, Israel), 1 mM sodium pyruvate (JHR Biosciences Industries, EU) and 10% heat-inactivated fetal bovine serum (Gibco Middlesex, England) was used as a complete medium for all cell cultures.

Antibodies The mAbs directed towards human CD57 (HNK1 and CD56 (HB55) were obtained from hybridoma producing mAb cells (American Type Culture Collection, Rockville, Madagascar). The kappa chain anti-mouse mAb labeled with PE was obtained from by Becton Dickindon (San José, California).

Cells Chinese hamster ovary (CHO) Kl cells were transfected with human cDNA encoding the CD28 gene in Pharmacia & Upjohn, Stockholm, Sweden. The transfected cells were routinely analyzed for CD28 expression and maintained by FACS which classify them at similar levels of antigenic expression. The human colon carcinoma cell line Colo205 was obtained from ATCC. All cell lines were free of mycoplasma. T-lymphocyte proliferation assessment. T cells were obtained from human peripheral blood mononuclear cells (PBM) as previously described (Lando et al 1996) by negative selection by taking with CD57mAbs, HLA-DR14 and CD56 . All tests on T cells were carried out with 0.1 x 10 cells / well in 200μm volumes, using 96-well flat bottom plates (Nunc, Roskilde, Denmark). DNA synthesis was studied after exposure of the cultures to [3H] -thymidine [JH] TdR (0.5mCi / well) as described previously (10).

Analysis by flow cytometry. Flow cytometric analysis and classification were carried out according to a standard location in a FACStar Plus flow cytometer (Becton Dickinson, Mountain View, California). Due to the low affinity that CD80 has for CD28, the staining of CHO-CD28 cells with the CD80-C2115Fab fusion proteins was omitted by washing the cells. Valuation of cytokines. The production of IL-2 was analyzed using an IL-2 ELISA kit (huIL-2 Duoset, Genzima).

Results Description of the fusion proteins CD80-Fab. The Fab portion is of a murine IgGl / k isotype although it contains the variable domains of the monoclonal antibody C215 IgG2A / k (Dohlstein et al 1995). The tripeptide sequence GGP follows the intermediate chain disulfide forming a cystine in the CH1 domain. In the triple fusion protein CD80-C215Fab-SEAm57, this functions as a separator between Fd and a mutant staphylococcus a-enterotoxin (SEA, Betley et al 1988), which has five substitutions. Replacements F47A and D227A were introduced to decrease the affinity of MHC class II (Abrahmsen et al 1995) and replacements N102Q, N149D and T218V were introduced to prevent fortuitous glycosylation when they occur in eukaryotic cells. These subsequent replacements are selected with the help of an X-ray structure (Sundstr [om et al 1996). The final pentamutant was designated mutant 57. Both fusion proteins contain the extracellular domain of human CD80 (defined for the termination of FPDN). The native CD80 signal peptide was used and the mature protein was found to start with HIVV as determined by an amino acid sequencing of purified CD80-C215Fab. An 18 amino acid separator connects CDO with the kappa chain in CD80-C215Fab-SEAm-57, or the Fd portion of the Fab fragment in the triple fusion protein CD80-C215Fab-SEAm57. The separators resemble a Q-linker (Wooton et al 1996) and have the sequences SARQANELPGAPSQEERA (SEQ ID NO: 15) and SARQANELPGAPSQEERP (SEQ ID NO: 16), respectively.

Facs Analysis: Binding of CD80-C215Fab fusion proteins to CD28 positive cells and to positive C215 cells.

CD28 positive cells. CHO-CD28 cells were stained with CD80-C215Fab, CD80-C215Fab-SEAm57 or C215Fab-SEA followed by an incubation with kappa chain anti-mouse mAb labeled with PE. The staining was carried out at 4 ° C and without washing. Both CD80-C215Fab and triple fusion proteins were bound to CD28 which is expressed for CHO cells in a dose-dependent manner. No staining with the C215Fab-SEA fusion protein was found as expected.

C215 positive cells. The agglutination of the fusion proteins against the C215 antigen was evaluated against the Colo205 cells. Colo205 cells were stained with CD80-C215Fab, CD80-C215Fab-SEAm57 or C215Fab-SEA followed by incubation with a PE-labeled anti-mouse kappa mAb chain. The staining was carried out at 4 ° C with three washes between each staining step.

Results (Figure 1-2): Both CD80-C215Fab and CD80-C215Fab-SEAm57 were agglutinated to Col6 205 cells positive for C215 antigen in a dose-dependent manner. The agglutination was 50-100 times lower than that for C215Fab-SEA: This indicates that the introduction of CD80 into the N-end portion of the fusion protein may interfere with the agglutination of the C215Fab part to the C215 antigen.

Double mergers Co-stimulation of T cells activated by a superantigen.

To determine the biological activity of the fusion proteins, these were checked for the co-stimulation of T cells activated by a superantigen.

Proliferation: T cells were incubated with Colo205 and 4uM C242Fab-SEA cells and varying amounts of CD80-C215Fab for 4 days after which they were quantified according to JH-thymidine. The activity obtained without any CD80-C215Fab was 20039 cpm +/- 1750.

Production of IL-2. T cells were incubated with Colo205 and 4uM C242Fab-SEA cells and varying amounts of CD80-C215Fab for 4 days after which the supernatant was harvested and the amount of IL-2 was determined. The amount of 11-2 was obtained without any CD80Fab-C215Fab and was 2849 pg / ml.

Results (Figures 3-4): CD80-C215Fab co-stimulated the induced activation of C242Fab-SEA T cells in a dose-dependent manner and both were seen with a proliferation and production of IL-2.

Triple fusions. Co-stimulation of T cells activated by an antigen.

To test the biological activity of the triple fusion proteins, the purified T cells were incubated with C215Fab-SEAm23 (m23 is the same SEA mutant that affects the agglutination of the MHC class II that was used in the triple fusion protein, ie Fe47Ala / Asp227Ala or CD80-C215Fab-SEAm57 present in Colo205 cells Proliferation: Incorporated -I-thymidine was counted after 4 days.Production of IL-2: the supernatants were collected and the content of IL-2 was determined of 4 days.

Results (figures 5-6). T cell activation induced by triple fusion proteins and IL-2 production was presented in Colo205 cells. No such activity was observed with C215Fab-SEAm23 indicating the importance of co-stimulation by CD80 activation. Due to the low agglutination affinity of triple fusion proteins (50-fold lower than with C215Fab-SEA, see FACS data), the current amount of C215Fab-SEAm23 bound to cells is likely to be substantially higher that one for the triple fusion proteins.

Example 2: IL-2 seeking, potentiates and prolongs the effects of FabSEA on the activation of T cells and tumor therapy.

Methods and materials: Construction of IL-2 expression plasmids. He IL-2 cDNA was cloned by RT-PCR using mRNA isolated from human peripheral blood mononuclear cells (PBM) which were stimulated with the superantigen SEA for 24 hours. The mRNA was isolated from 5xlOb cells using a set of Direct mRNA implements from Dynal, Oslo according to the manufacturer's instructions. An elution was made to the magnetic beads with an mRNA envelope bound to an oligo (dT) 26 by heating at 95 ° C. Subsequently, a PCR product was obtained by TR-PCR, taking advantage of the activities of the DNA polymerase and RT of the Tae polymerase. 1/10 of the m RNA was eluted with OCR arbors IL2-1 and IL2-2 and a standard PCR reaction (30 cycles) was carried out. The PCR product was subjected to an agarose gel electrophoresis, the band was excised from the gel and purified using a set of Prep-a-gene implements and a set of implements (Bio-Rad). After digestion with EcoRI or BamHI, the fragment was cloned in EcoRI / BamHI from p photocalca digested KS II. the incersion was sequenced and confirmed to be identical to the previously reported IL-2 cDNA (Taniguchi et al 1983).

However, as a result of a PCR reaction, a segment of DNA encoding Gly-Pro-Arg-Gln-Ala-Asn-Glu-Leu-Pro-Gly-Ala-Pro-Ser has been added to 5 '. -Gln-Glu-Glu-Arg (SEQ ID NO: 23) "Gly-Pro-Q linker" to the segment encoding human adult IL-2. A cloning of Q-hIL2 in a plasmid was made from a domestic collection (which is cut with Rsrll-Xbal) as an RsrII-Nhel fragment. The resulting plasmid, pMS306, directs the secretion of C215Fab SEA-Q-hIL2 to the periplasm of E. coli. Porters of the portions were further optimized as follows. A reaction encoding a linker, coding for Pro-Ala-Ser-Gly-Gly-Gly-Gly-Ala-Gly-Gly-Pro 8SEQ ID NO: 19) (replacing the original Gly-Gly-Pro) was introduced between the superantigen and the regions that code for a Fab portion using site-controlled mutagenesis. Similarly, the linkers Gly-Pro-Arg-Gln-Ser-Asn-Glu-Tr-Pro-Gly-Ser-Pro-Ser-Gln-Glu-Glu-Arg- (SEQ ID NO: 20), Gly- Pro-Arg-Gln-Ala-Lis-Tr-Leu-Pro-Gly-Ala-Pro-Ser-Gln-Tr-Tr-Arg (SEQ ID NO: 21) or Gly-Pro-Tr-Glu-Ala.Asp -Glu-Leu-Pro-Gly-Ala-Pro-Ser-Glu-Glu-Glu-Tr (SEQ ID NO: 22) replaced the original Q linker between IL-2 and the Fab portion. For combinations of double fusion proteins, structures with IL-2 were also fused to the heavy ligand chain respectively, where they were compared.

Primary primers IL2-1: 5 '-GCG GAT CCC GGT CGT CAG GCT AAC GAA CTG CCA GGA GCT CCG TCT CAG GAA GAG CGT GCA CCT AC TTC AAG TTC TAC AAA G-3 '(SEQ ID NO: 17) IL2-2: 5' -CCG AAT TCG CTA GCT TAT CAA GTT AGT GTT GAG ATG AT-3 'ÍID SEQ NO: 18) Expression of the fusion proteins in the fermenter. The fusion proteins were expressed in strain K-12 of E. coli UL 635 (xyl-7, ara-14 T4R, deltaompT) using a plasmid with a kanamycin resistance gene and a lacUV5 primer. Bacteria from a frozen variety were incubated at 25 ° C for approximately 21 hours in flasks inside a vibrating screen containing (g / 1) (NH4) 2S04, 2.5; KH2P04, 4.-5; 'K2HP04, 11.85; sodium citrate, 0.5; MgSOj .7H20, 1; glucose monohydrate, ll, 0.11 mM kanamycin and 1 ml / l trace element solution (Forsberg et al., 1989), however without Na2Mo04.2H20. the cells were grown to OD600 of 1-2 and 450ml of culture medium was used to inoculate a fermentor (Chemap, Switzerland) to a final volume of 51. the medium of the fermenter contains (g / 1) (NH4) 2S0, 2.5; KH2P04, 9; K2HP04, 6; sodium citrate, 0.5; glucose monohydrate, 22, O.llmM of kanamycin and 1 ml / l MgSO4.7H20, 1; 1 ml of adecanol (Asahi Denka Kogyo K. K, Japan) and 1 ml / l of trace element solution. The pH was maintained at 7.0 by means of a titration with 25% ammonia, the temperature is 25 ° C and the atmospheric air aeration of 5 l / min. The dissolved partial pressure of 02 is controlled to 30% by an increase in agitation of 300 to 1000 rpm during the batch phase and by regulating the nutrients of 60% (w / v) glucose during the batch-fed phase. The formation of the product was induced in an ODgoo of 50 by adding mM of isopropyl β-D-thiogalactopyranoside, IPTG. After fermentation, the cells were removed by centrifugation at 8000 xg for 40 min at 4 ° C. The improved medium was analyzed and purified directly or stored at -20 ° C.

Purification of fusion proteins. The DNA present in the purification medium was removed using a precipitate with 0.19% polyethylenimine and 0.2M NaCl for 30 min (Atkinson and Jack, 1973). After the previous centrifugation, the supernatant was collected and the concentration of NaCl was adjusted to 0.5M. this medium was applied to a Protein G Sepharose column (Pahrmacia Biotech AB, Uppsala, Sweden) and equilibrated with lOmM sodium phosphate, 150 mM NaCl, pH 7.4 containing 0.05% Tween 80, PBST. The column was then washed with 5 volumes of PBST column and elution of the agglutinated protein was made with 0. IM acetic acid, 0.02% Tween 80 pH 3.2. the pH of the sample was adjusted to 5.0 using 1 M Tris-HCl, pH 8.0 and applied to an HP Sepharose SP column (Pharmacia Biotech) and equilibrated with 50 mM ammonium acetate, 0.02% Tween 80. Then the column was washed with 2 column volumes of an equilibrium buffer and elution of the fusion protein was done using a linear gradient from 50 to 500 mM of ammonium acetate over 10 column volumes. Given the c215Fab-IL2 fusion proteins, a pH of 6.0 was used while the triple fusion proteins C215Fab-SEA-IL2, the pH was 5.7 during the separation. The fusion proteins were filtered through a 0.22 μm filter and stored at -70 ° C. If diluted further, the eluto is concentrated to a final concentration of 0.5-1 mg / ml using Centricon 30 (Amicon) according to the manufacturer's instructions.

Valuations of cytotoxicity. Titrations in cytotoxicity for MHC class II dependent and independent as previously described (Dohlsten et al., 1990). Briefly, for MHC class II dependent assays, Raj i cells labeled with xCr (2500 cells per well in a final volume of 200 ul) were mixed in an SEA-dependent effector cell line by incubation of human PBM in the presence of low levels of recombinant hIL-2. Effector: The target cell was 30: 1. For the MHC Class II independent assays, Colo205 (2500) C215 + cells labeled with 51 Cr were incubated with SEA-dependent effector cells at an E: T ratio of 45: 1. The 51Cr released in the medium was determined after 4 hours of incubation after the scintillation count.

In vitro evaluation of the co-stimulation of purified human T cells. Natural human T cells were purified in an essential manner as described by Lando et al. (1993). Briefly, the natural human T cells were purified from a PBM of human blood by a centrifugation of Ficol gradients, followed by a separation of gelatin columns, and finally a negative selection by a take in the Petri dishes that contain HNK1 and HLA-DR mAbs. Proliferation experiments using natural human T cells were carried out as described by Lando et al (1996). Briefly, natural T cells (10,000 cells per well) were combined by transfectants irradiated by CHO (10,000 cells per well) in a total volume of 200 ul RPMI-1640 with supplements (Lando et al 1993). For experiments with IL-2 containing fusion proteins, the cells were incubated for 7 days in the presence of InM of the indicated substances. On the last day of the experiment the cells were pulsed with 3H-thymidine to quantify the DNA incorporation of the dividing cells.

Tumor therapy B16-C215. The therapy was carried out in essence, as previously described (Dohlstein et al 1994, Hansson et al 1997). On day 0, C57B1 / 6 mice were injected. v. in the vein of the tail with 75,000-150000 B16-F10 syngeneic melanoma cells. These Bl cells were expressing for the human GA-733 antigen recognized by the C215 mAb. On day 1,3 or 5 he started therapy with C215Fab proteins. On day 21 the experiment was finished, at that time the lungs were removed and the disseminated tumors of the lung were counted.

Immunohistochemistry It was carried out in the lungs of animals carrying tumors B16-C215 of Day 18 in essence, as previously described (Dohlsten et al 1995). The samples were removed 4 hours after the final injection. The stained area was determined manually.

Immunopharmacology It was carried out essentially as described (Rosendahl et al 1996). The vessels of C57 Bl / 6 mice that received 1 or 3 injections, once a day, were removed 48 hours after the final injection and the SEA-dependent cytotoxicity was determined, using target Raji cells, in a standard assessment. of 51Cr release as described above. The E: T ratio was 100: 1.

Results and Discussion Production and purification of fusion proteins containing IL-2. A vector expressing for E. coli and coding for a triple fusion protein C215FabSEA-Q-hIL2 was constructed. This vector codes for two subunits of the triple fusion protein in a biscistronic mRNA that are transcribed from the LacUV5 promoter. Each of the two subunits is received by a signal peptide which controls an export to the periplasmic space of E. coli. the first subunit is VH of the C215Fab followed by a Gly-Gly-Pro linker (VH-CHl-Gly-Gly-Pro-SEA). The other subunit is VK from C215Fab followed by CK of C242Fab, which is linked to human IL2 by a Gly-Pro-Q linker (VK-Ck-Gly-Pro-Q-hIL2) . The sequence of the Gly-Pro-Q linker (SEQ ID NO: 23) is a slightly modified version of a natural linker found in the OmpR protein of E. coli (Wootton et al 1989). See materials and methods for more complete information. A C215FabSEA-Q-hIL2 fusion protein was also produced. It is identical to C215FabSEA-Q-hIL2 except that the Gly-Gly-Pro-SEA portion of the protein has been removed. The corresponding DNA sequence in the expression vector is deleted accordingly. Mutated derivatives of C215FabSEA-Q-IL2 have been generated by a site-directed mutagenesis at the PCR-mediated site using standard methods to produce several C215FabSEA-like proteins D227A-Q-hIL2 and C215Fab SEA D227A-Q-hIL2F242A. In later variants of the triple fusion protin the intersubunit cystine is replaced by the serine residues. This alteration does not affect the biological activity.

The plasmids encoding the IL-2 containing proteins were transformed into the E. coli UL635 production strain, and subsequently the fermentation was carried out. The fusion proteins were purified from the culture medium using affinity chromatography for Protein G. The degraded variants of the fusion proteins were removed using ion exchange chromatography. The points obtained were at least 90% total length fusion proteins; as determined by SDS-PAGE (material and methods). Using the optimal design of the fusion protein, up to 13Omp / 1 fusion proteins is obtained in the growth medium and typically 70 mg of fusion proteins were obtained from 1 liter of the medium.

Functional characterization of Fab fusion proteins containing IL-2. The ability of fusion proteins containing IL-2 such as C215FabSEA-Q-hIL2 and C215Fab-Q-hIL2 fusion proteins to induce proliferation of the murine CTLL-2 cell line dependent on IL-2 was essentially similar to that of recombinant human IL-2 on a molar basis (data not shown). On the other hand, the agglutination of antigens and the SEA activity of C215FabSEA-Q-hIL2 and C215FabSEA was found to be indistinguishable in several titrations, suggesting that there are no adverse effects of IL-2 in a molecule. These titrations have the ability to induce, on the part of the MHC class II, an independent annihilation of the human colon cancer cell line C215 + by a T cell effector cell line and reactive SEA salt during a 4 hour release of 5iCr. Also the dependent annihilation by the MHC class II + of the Raji cells (rat lymphoma) by effector T cells reactive to the SEA proceeds with similar efficiencies (data not shown). The most direct evidence for the uncommitted C215 antigen and the agglutination of MHC class II was obtained by FACS analysis. The dose dependence of C215FabSEA-Q-hIL2 and C215Fab-Q-hIL2 that agglutinate the cells of Ra i that was similar to a molar base (not shown) and C215Fabn-Q.hIL2 to the Colo205 cells was found to be similar to that observed for C215FabSEA (data not shown) and indicating that antigen agglutination was not compromised in the fusion proteins containing IL-2.

Proliferation induced by FabSEA dependent on IL-2. Resting human T cells require both a signal 1 and a signal 2 to drive the activation and optimal proliferation of T cells (Schwartz, 1990). Superantigens can send signal 1, if present in a cell. IL-2 is the main effector of the 3 'direction of B7 / CD28 signaling is expected to produce signal 2.

Here we show that using resting T cells purified from human blood that a triple fusion protein C215FabSEA-Q-hIL2 or a C215FabSEA in combination with C215Fab-Q-hIL2 or recombinant human IL-2 certainly does not induce a proliferation of T cells in vitro (figure 7).

Resting human T cells were incubated with target SEA (C215FabSEA or C215FabSEA-Q-hIL2) in the presence of IL-2 (in the form of C215Fab-Q-hIL2, C215FabSEA or recombinant human IL-2). The SEA was presented in irradiated CHO cells transfected with the C215 antigen (via the Fab portion), MHC class II / DR that binds to the SEA. The non-transfected CHO cells served as a control. After 7 days of incubation of the T cells with the CHO transfectants and the substances in question, the proliferation was quantified by the incorporation of 3H-thymidine into the DNA.

C215FabSEA-Q-hIL2 induced the proliferation of human T cells when presented in other CHO cells transfected with the human C215 antigen for which the Fab was directed against (figure7). Also, proliferation was induced when the protein was presented in CHO-Dr (human MHC class II), considering that no proliferation was observed in the presence of non-transferred CHO cells (CHO) or in the absence of CHO cells (RIO). C215FabSEA or 'C215Fab-Q-hIL2 did not induce any significant proliferation by themselves when presented in CHO cells indicating that both SEA and IL-2 are indeed necessary for induction and proliferation. This was confirmed, by way of the combination of C215FabSEA and C215Fab-Q-hIL2 or C215FabSEA and recombinant human IL-2 induced an effect qualitatively similar to that induced by C215FabSEA-Q-hIL2. This also shows that in this assessment IL-2 is necessary but unlike SEA, the cell does not need to be bound.

C215FabSEA-Q-hIL2 as well as the combination of C215FabSEA and C215Fab-Q-hIL2 causes enhanced and sustained activation of T cells and improved tumor infiltration in vivo. Both C215Fab-Q-hIL2 + C215FabSEA, and C215FabSEA-Q-hIL2 were much more potent inducers of T cell activation than C215FabSEA as quantified by the ability to induce SEA-dependent annihilation of target cells (Figure 8). ). Briefly, the mice received 1 or 3 injections administered dariamente of the indicated proteins. Two days after the last injection, the vessels of the treated mice were removed and the cytotoxic activity was determined against the Raji cells coated by SEA in a standard 51 Cr release for 4 hours.

Both C215FabSEA-Q-hIL2 and C215FabSEA / C215Fab-Q-hIL2 induced not only an enhanced but also sustained activation of T cells. Serum cytokine levels such as IFNgamma., Which drastically sinks after the fourth injection in C215Fab ' SEA is maintained at a very high level even after the fourth injection of C215FabSEA-QhIL2 (data not shown). In general, IFNgamma and TNFalpha levels were much higher (up to 10 times higher) than in the case of C215FabSEA.

Improved therapy of B16-C215 tumors established in a combination treatment with C215FabSEA and C215Fab-Q-hIL2. The effects of the potentiation of IL-2 for FabSag tumor therapy were investigated in the murine melanoma model B16 (Figure 9). In the example shown, 8-week-old C57 Bl / 6 female mice were inoculated on day 0 with 150,000 B16 cells transfected with the human tumor GA-733 antigen, for which the C215 mAb is directed against ( B16-C215 in the subsequent). The indicated substances were injected on days 5, 6 and 7. Each information point corresponds to 7 animals. On day 21 the animals were sacrificed, and the number of B16 tumors pigmented with melanin and quer colonized the lung was counted. The combination of C215FabSEA and C215Fab-Q-hIL2 in several experiments was shown to induce better therapy than C215Fab SEA compared to a control without treatment (Figure 9), in the indicated experiment, the combined treatment of C215FabSEA / C215Fab-Q-hIL2 gave a better therapeutic effect than the triple fusion protein C215FabSEA-Q-hIL2.

Subsequent immunohistochemistry studies revealed that C215FabSEA alone or in combination with C215FabSRA and C215Fab-Q-hIL2 gave rise to similar numbers of CD4 and CD8 T infiltrating B16-C215 tumor cells. Interestingly, however, the number of CD25 positive cells (IL2Ra) - a good marker for T cell activation - increased dramatically between the third and fourth injection of 'C215FabSEA / C215Fab-Q-hIL2 (Figure 10). In contrast, it decreased in the case of C215FabSEA indicating the principle of anergy. The quality of infiltrating T cells in this way seems to be higher in the case of combination than in C215FabSEA alone, which may help to explain the improved therapeutic effect. Similarly, serum cytokines such as Interferon? secondary to the activation of T cells decreased markedly after the fourth injection of FabSEA (Figure 11). With a triple fusion protein FabSea-Q-IL2, however, the levels of Inferieron remain at a high level even after the fourth injection. This observation provides an additional indication that including 11-2 in the structure can counteract the anergy of T cells induced by Fab-SRA.

Improved tumor therapy B16-C215 with C215FabSEAD227A-Q-hIL2. Superantigens are much more toxic in humans than in mice, in part because of the affinity for MHC class II which is considerably higher (Hanson et al 1997). The systemic toxicity of FabSEA proteins is therefore anticipated to await the greatest limitation of therapy in humans. One way to increase local activation in the tumor (Fab-dependent) against systemic immune activation (dependent on SEA-MHC class II) would be to decrease the affinity of SEA for the MHC class II. based on the crystal structure of the SEA we made a mutation of C215FabSEA, C215FabSEAD227A / with a 100 fold lower affinity for MHC class II. unlike C215Fab-SEA it does not have the ability to cross-link MHC class II molecules, which is believed to be a major reason for systemic toxicity surrounded by SEA (Hanson et al 1997).

The window between the efficient window and the toxicity in the treatment of B16-C215 tumors from day 1 in transgenic Vb3 mice was at least 50 times wider than for the C215FabSEAD .._ 7A mutant compared to C215FabSEA (Hanson et al 1997) . Consistent with this observation, immunohistochemistry revealed that at doses of the two proteins that resulted in similar therapy, a comparable immune activation of the tumor was observed, whereas a much less systemic immune activation was observed in the spleen with C215FabSEAD -1A (Hanson et al 1997). Moreover, pharmacokinetic studies in rabbits showed a dramatic reduction in the transport of C215FabSEAD_.27A looking for, towards the spleen and other lymphoid organs, where the majority of MHC class II + cells are located, compared to C215FabSEA (not the data is displayed).

For clinical use it is expected that a triple fusion protein Fab-SEA-IL2 contains a superantigen that has been mutated. We therefore made a triple fusion protein C215FabSEAD227A-Q-hIL2. The protein produced was able to induce the proliferation of resting human T cells (data not shown) - and induced a CTL activity of sustained SEA slope in mice up to 6 injections (Figure 12). In contrast, background CTL activity was observed with C215FabSEAD227A (< 10% - data not shown) and C215Fab-hIL2 (< 15% - data not shown). Therapy for established tumors (day 3) B16-GA733 in normal C57 Bl / 6 mice with 8 injections of this protein administered daily gave good therapy with an optimal dose of C215FabSEA (Figure 13). In this system, C215FabSEAD227A, which is defined for optimal activity in humans, not mice, not only has minor effects (Figure 13). At the highest dose, some toxicity C215FabSEAD_-7A-Q-hIL2 was found, however. In contrast, several therapeutic experiments indicate that a combination of C215FabSEAD-: 27A and C215Fab-Q-hIL2 (8 injections) at least leads to a substantially improved therapy of B16-GA733 tumors on day 3 in transgenic Vb3 TCR mice when compared to C215FabSEAD227A only (data not shown). In transgenic mice Vb3 TGR > 90% of T cells react with SEA, as opposed to 10-20% in normal mice. Interestingly, in rabbits the repeated injections (up to 8) of C215FabSEAD227A -Q-hIL2 (0/4 dead rabbits after repeated injections at 20 μg / kg) do not appear to be more toxic than C215FabSEA without IL-2 (0 / 4 dead rabbits after repeated injections at 20μg / kg, 4/4 dead at 20μg / kg) much less toxic than C215FabSEA (1/4 rabbits killed after repeated injections at 1μg / kg). At the same time, a sustained immune activation (lymphocyte rebound effect) was observed only after treatment with C215FabSEAD227A-Q_hIL2 but not with C215FabSEAD227A or C215FabSEA - indicating that in fact the inclusion of IL-2 helps counteract induced T cell anergy by FabSEA (data not shown).

Tumor therapy B16-C215 with C215FabSEAD227A-Q-hIL2 proteins that have mutated IL-2. The greater toxicity, in the case of C215FabSea-Q-hIL2 a lower therapeutic efficacy, of triple fusion proteins containing IL-2 may in part be due to "re-search towards the target" of SEA activity in lymphoid tissues . This re-search effect towards the target is due to the high affinity of IL-2 (Kd = 10 pM) for its receptor, which is found mainly in the T cells in the spleen and in other lymphoid tissues. To discuss this issue we have made derivatives of where the IL2 portion has been mutated in order to reduce the affinity for IL2R cells "both their systemic activation.In principle, the same approach that was used to improve the therapeutic window for the potency C215FabSEAD227A- The interaction between IL-2 and its high affinity receptor is very well studied. The receiver consists of 3 subunits a, b and g. Cross-linking of b and g is required for biological activity, while a subunit is required for optimal binding. Our strategy is to reduce the affinity of IL2 for its high affinity receptors (abg) by eliminating the agglutination of the subunit a, and subsequently reducing but not eliminating, the agglutination of subunits g and b as necessary.

Three similar mutants of C215FabSEAD2: 7A-Q-hIL2 were designed to eliminate the binding of IL2Ra (F42A, F42K) and also impart the IL2Rb binding (F42A / D20S). These proteins have 100- (F42A), 1000- (F42K) and 3000 times (F42A / D20S) of reduced activity, respectively, in inducing the proliferation of murine CTLL-2 cell line dependent on IL-2. There are experiments in progress to determine in greater detail the properties of these proteins, in particular with respect to affinity and in the proportions for the binding of human T cells and the IL-2 receptor. Initial therapy experiments indicate that such a strategy is promising (Figure 14, Figure 15). In the examples shown, 8 injections were given to transgenic Vb3 TCR mice (where more than 90% of the T cells were activated by SEA) carrying B16-GA733 tumors from day 3. Although some toxicity is still observed at the higher dose and not later when the C215FabSEAD227A-Q-IL2F42A or C215FabSEAD227A-Q-IL2F-2? (8a.Injection) instead of C215FabS_AD227A-Q-IL2 was used (6th injection) and treatment at the highest dose consistently with an SEA greater than 90% tumor reduction when compared to the control treated with PBS (Fig. 14). We are currently exploring this highly promising approach by introducing additional mutations in the SEA and parts of IL2 to further improve therapy for the toxicity window.

Furthermore, there is work in progress to make the triple fusion proteins C215FabSEA-Q-hIL2 comprising a Tr51Pro mutation in the IL-2 part (Chang et al 1996). This mutation can serve to block the IL2R-mediated internalization of the fusion proteins without reducing the bioactivity of IL-2. This is likely to be important as it will reduce the extraction of the fusion protein by this route, and thereby increase the local concentration and efficiency of the drug. The proteins that Tr5Pro mutated may contain more mutations in the IL-2 and SEA parts to reduce affinity for the MHC class II and the IL-2 receptor, respectively.

Example 3. Experiments to verify the effects of the conjugates (Sag, IM). Positive cells for the receptor -IM or cells positive for the MHC class II.

A Sag-IM molecule can bind to the cells that express MHC class II, thereby facilitating the SDCC. Alternatively, cells expressing for the IM receptor can go to the target. It is therefore possible that a Sag-IM molecule could be useful for inducing the annihilation of undesirable cells expressing for the IM receptor.

A triple fusion protein C215FabSEA-Q-hIL2 can be considered a Sag-IM molecule in the event that no effector or target cell expresses for the antigen recognized by C215Fab. The effector cells are T cells of the correct subtype Vß. The target cells can, i.e., be aberrant hematopoietic cells expressing for the IL-2 receptor similar to those present in certain leukemias or lympholas.

Experiment A: Demonstration of the concept in vi tro: effector T cells (human T cells stimulated repeatedly with SEA) and target cells (e.g., B-cell lymphoma cells) "Raji") human) will be incubated with the triple fusion protein C215FabSEAhIL2 that has been mutated to reduce its agglutination to the MHC class I. This is the standard mechanism for titrations SDCC (effector T cells, Raji cells, experimental substance). We have now found that the C215FabSEAD227A-hIL2 protein with a severely reduced affinity for the MHC class II in approximately 10 times more potency than C215FabSEAD, 27A in the annihilation of Raji cells. An obvious explanation for the increased potency is that the triple fusion protein occurs at the IL2 receptors expressed for Raji cells.

Experiment B: Demonstration of the in vi vo concept: Murine lymphoma models (such as RBL-5 lymphoma in C57 Bl / 6 mice) are well established (Hoglund et al J Exp Med 168, 1469-1474). The search for the IL-2 receptor or another IM-R with a Sag-IM protein in such models can provide a demonstration of the concept for the target cells positive for the IM receptor in vivo.

The Sag-IM that looks for cells positive to IM-R is complicated by the fact that both effectors and target cells frequently express for the IM receptor. However, under certain circumstances this mode of therapy can be effective. Factors such as density of IM-R expression in target cells, a location of target cells, etc., are likely to play a role. It should also be emphasized that, for example, aIL2-R is only over-regulated at the time of T-cell activation - perhaps in this way there may be a time window during which IM-R is expressed in high abundance for target cells, but not on the effector cells.

The Sag-IM fusion proteins in which Sag symbolizes a wild-type antigen that has been mutated for reduced affinity to the MHC class II can be used in a similar manner.

REFERENCES Marked articles are * are more related to immunomodulators than others.

Abrahmsén L et al (1995) Characterization of two distinct MHC Class II binding sites in the superantingen staphylococcal enterotoxin A. EMBO J 14: 2978-86.

Abrahmsén et al (1996) W09601650 (patent applicattion). To conjugate betwen to modified superantigen and a targetseeking compound and the use of the conjugate.

Antonsson P et al (1996) Staphylococcal enterotoxin A and E chimera with reduced seroreactivity and retained ability to target cytotoxic T cells for use in tumor therapy. ABRF '96: Biomolecular Techniques, Holiday Inn Golden Gateway, San Francisco, California. March 30 -April 2, 1996.

Antonsson et al (1996) J. Immunol. 158: 4245 4251.

Antonsson et al (1997) WO 976932 (patent application) Atkinson et al (1973) Biochim. Biophys. Acta 308: 41-52.

* Bamboroug et al. (1994) Structure 2: 839- * Becker et al (1996) Eradication of human hepatic and pulmonary melanoma metastases in SCID mice by antibody interleukin 2 fusion proteins. Proc. Nati Acad. Sci. USA 93: 2702-2707.

* Belfrage Thesis (1996) Activation of murine cytotoxic cells with interleukin 2 and the bacterial superantigen stapylococcal enterotoxin A. University of Lund. Sweden (Augusti / September 1996).

* Belfrage et al (1994) Combined activation of murine lymphocytes with staphylococcal enterotoxin and interleukin 2 results in additive cytotoxic activity. Cancer Immunol. Immunother 38: 265-271).

* Belfrage et al (1995) Enhanced and prolonged efficacy of superantigen induced cytotoxic T lymphocyte activity by IL-2 in vivo. Cancer Immunol. Immunother. 41: 87-94.

* Belfrage et al (1997a) Prevention of superantigen induced downregulation of T cell mediated cytotoxic activity by IL-2 in vivo. Immunology 90: 183-188.

* Belfrage et al (1997b) Prevention of superantigen induced tolerance in vivo by IL-2 treatment. Submitted 1996.

* Berndt et al (1994) Biochemistry 33: 6571-, Betley et al (1988) Nucleotide sequence of type A staphylococcal enterotoxin gene. J. Bacteriol. 170: 34-41.

Boyer et al (1969) A complementation analysis of the restriction and modification of DNA in Escherichia coli. J.Mol. Biol. 41: 459-472.

Blanco et al (1990) Mutants of staphylococcal toxic shock syndrome toxin 1: Mitogenicity and recognition by a neutralizing monoclonal antibody. Infect. Immun. 58 (1990) 3020-3028.

Carlsson et al (1985) Kinetics of IL-2 and Interferon gamma production, expression of IL-2 receptors, and cell proliferation in human mononuclear cells exposed to stap? Ylococcal enterotoxin A. Cell. Immunol. 96: 175-183.

* Chen et al (1992) Costimulation of antitumor immunity by the B7 receptor for the T lymphocyte molecules CD28 and CTLA. Cell 71: 1093-102.

* Chang et al (1996) to point mutation in Interleukin-2 that alters ligand internalization. J. Biol. Chem. 271: 13349-13355.

* Collins et al (1988) Proc. Nati Acad. Sci. USA 85: 7709-.

Dohlsten et al (1988) Two subsets of human blood CD4 + T helper cells differing in the capacity to produce 1L-2 and interferon-gamma can be defined by the Leu-18 and monoclonal antibodies. Eur. J. Immunol. 18: 1173-1178.

Dohlsten et al (1990) Im unology 71: 96-100.

Dohlsten M et al (1991) Monoclonal antibody-targeted superantigens: A different class of antitumor agents. Proc. Nati Acad. Sci. USA 88: 9287-9291.

Dohlsten et al (1992) W09201470 (patent application). Target specific antibody superantigen conjugates and their preparation.

Dohlsten M et al (1994) Monoclonal antibody-superantigen fusion proteins: Tumor specific agents for T cell bassed tumor therapy. Proc. Nati Sci. USA 91: 8945-8949.

Dohlsten et al (1995) Proc. Nati Acad. Sci. U.S.A. 92: 9791-9795.

* Dow et al (1996) W09636366 (patent application) Combined gene therapy with the gene for a superantigen and the gene for a cytokine or chemokine.

* Fell et al EP 439095 (Patent Application) Fleury S et al (1991) Mutational analisys of the interaction between CD4 and class II MHC: class II antigens. Cell 66: 1037-49.

Forsberg G et al (1989) BioFactors, 2, 105-112.

Hansson J et al (1997) Proc. Nati Acad. Sci. -U.S.A. 94 (i? Press).

Kalland et al (1991) WO 9104053 (patent application). Pharmaceutical composition that makes cells expressing MHC Class II antigens targets for cytotoxic cells.

Kappler JW et al (1992) Mutations defining functional regions of the superantigen staphylococcal enterotoxin B. J. Exp. Med.175: 387-396.

Kappler et al (1993) W09314634 (patent application). Protective efects of mutated superantigens.

Kotzin BL et al (1993) Superantigens and their potential role in human disease. Adv. Immunol. 54: 99-166.

Lamphear JG et al (1996) Residues near the amino and carboxy terminal of staphylococcal enterotoxin E independently mediated TCRWβ specific interactions. J. Immunol. 156: 2178-2185 (March 15,1996).

* Lando PA et al (1993) Co-sti ulation with B7 and targeted superantigen is required for MHC class II independent T cell proliferation but not cytotoxicity. Im unology 80: 26-241.

* Lando et al (1996) Regulation of superantigen induced T cell activation in the absence and the presence of MHC class 11. J. Im unol. 157: 2857-2863 Lindholm et al (1993) 9301302 (patent application). Tumor, carbohydrate antigen specific monoclonal antibody and cell line.

Newel et al (1991) In vivo T-cell activation by staphylococcal enterotoxin B prevents outgrowth of a malignant tumor. Proc. Nati Acad. Sci. USA 88: 1074-1078.

Ochi et al (1993) J. Immunol. 151: 3180-3186.

* Pancook et al (1996) Eradication of established hepatic human neuroblastoma metastases in mice with severe combined immunodeficiency by antibody targeted interleukin-2. Cancer Immunol. Immunother. 42: 88-92.

* Pouletty (1993) EP 510949 (patent aplication) Targeted cytolysis.

* Rosenblum et al EP 396387 (patent application).

* Rosendahl et al (1996) Int. J. Cancer 68, 109-113.

Sanger et al (1977) DNA sequencing with chain-terminating inhibitors. Proc. Nati Acad. Sci. USA 74: 5463-5467, 1977.

Sambrook et al (1989) Molecular Cloning. A laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press.

* Schwartz (1990) Science 248: 1349-1356.

Stern et al (1989) W08907947 (patent application). Improvements relating to antigens.

Sundstrom et al (1996) J. Biol. Chem. 271: 32212-32216.

* Taniguchi et al (1983) Structure and expression of a cloned cDNA for human Interleukin-2. Nature 302: 305-310. * Terman et al (1991) W09110680 (patent application) Tumor killing effets of enterotoxins and related compounds.

* Terman et al (1993) W09324136 (patent application) Tumor 'killing effets of enterotoxins and related compounds.

Woodworth (1993) Preclinical and Clinical Development of Cytokine toxins presented at the conference "Molecular Approaches to Cancer Immunotherapy", Ashville, North Carolina, November 7-11, 1993.

Wootton et al (1989) Prot. Eng. 2: 535-543. * Zurawski et al (1993) EMBO J. 12: 5113-.

LIST OF SEQUENCES (1) GENERAL INFORMATION: (i) APPLICANT: (A) NAME: Pharmacia & Upjohn AB (B STREET: Lindhagensgatan 133 (C) CITY: Stool or (E) COUNTRY: Sweden (F) ZIP CODE (ZIP): S-112 87 (G) TELEPHONE: +46 8 695 80 00 (ii) TITLE OF THE INVENTION: Directed cytolysis of target cells, agents and compositions that cause cytolysis, and combinations that can be used to produce the agents. (iii) SEQUENCE NUMBER: 23 (iv) LEGIBLE FORM OF COMPUTE (A) TYPE OF MEDIUM: Flexible disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) NON-PHYSICAL COMPONENTS: Patent in Version No, 1.0 , Version No. 1.30 (EPO) (2) INFORMATION FOR THE IDENTIFICATION OF SEQUENCE No. 1: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 33 base pairs (B) TYPE: Nucleic acid (C) No OF HEBRAS: A (D) TOPOLOGY: Linear (ii) TYPE OF MOLECULE: Other nucleic acid (A) DESCRIPTION: / desc = "primer of the DNA oligonucleotide chain" (xi) DESCRIPTION OF THE SEQUENCE: IDENTIFICATION OF THE SEQUENCE No: 1: ATATAAGCTT CCACCATGGG CCACACACGG AGG 33 (2) INFORMATION FOR THE IDENTIFICATION OF SEQUENCE No. 2: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 35 base pairs (B) TYPE: Nucleic acid (C) No. OF HEBRAS: One (D) TOPOLOGY: Linear (ii) TYPE OF MOLECULE: Other nucleic acid (A) DESCRIPTION: / desc - "primer of the DNA oligonucleotide chain" (xi) SEQUENCE DESCRIPTION: IDENTIFICATION OF THE SEQUENCE No: 2: ACGCAGATCT TTAGTTATCA GGAAAATGCT CTTGC 35 (2) INFORMATION FOR THE IDENTIFICATION OF SEQUENCE No. 3: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 39 base pairs (B) TYPE: Nucleic acid (C) No. OF HEBRAS: One (D) TOPOLOGY: Linear (ii) TYPE OF MOLECULE: Other nucleic acid (A) DESCRIPTION: / desc = "primer of the DNA oligonucleotide chain" (xi) DESCRIPTION OF THE SEQUENCE: IDENTIFICATION OF THE SEQUENCE No: 3: TCAAAGCTTC TCGAGCGCGC TGTTATCAGG AAAATGCTC 39 (2) INFORMATION FOR THE IDENTIFICATION OF SEQUENCE No. 4 (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 46 base pairs (B) TYPE: Nucleic acid (C) No. OF HEBRAS: One (D) TOPOLOGY : Linear (ii) TYPE OF MOLECULE: Other Nucleic Acid (A) DESCRIPTION: / desc = "Sequence primer of DNA oligonucleotides (xi) DESCRIPTION OF THE SEQUENCE: IDENTIFICATION OF THE SEQUENCE No: 4: CGCGCGTCAG CGTAACGAAC TGCCAGGCGC CCCGTCACAG AGACG 6 (2) INFORMATION FOR THE IDENTIFICATION OF SEQUENCE No. 5: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 60 base pairs (B) TYPE: Nucleic acid (C) No. OF HEBRAS: One (D) TOPOLOGY: Linear (iii) TYPE OF MOLECULE: Other nucleic acid (B) DESCRIPTION: / desc = "primer of the DNA oligonucleotide chain" (xi) DESCRIPTION OF THE SEQUENCE: IDENTIFICATION OF THE SEQUENCE No: 5: AGCTTCGTCT CACGCGCGTT CTTCCTGTGA CGGGGCGCC GGCAGTTCGT TAGCCTGACG 60 (2) INFORMATION FOR THE IDENTIFICATION OF SEQUENCE No. 6: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 32 base pairs (B) TYPE: Nucleic acid (C) No. OF HEBRAS: One (D) TOPOLOGY: Linear (ii) TYPE OF MOLECULE: Other nucleic acid (A) DESCRIPTION: / desc = "primer of the base of DNA oligonucleotide" (xi) DESCRIPTION OF THE SEQUENCE: IDENTIFICATION OF SEQUENCE No. 6: TGGTACACCA CAGAAGACAG CTTGTATGTATG 32 (2) INFORMATION FOR THE IDENTIFICATION OF SEQUENCE No. 7 (iv) CHARACTERISTICS OF THE SEQUENCE: (A). LENGTH: 32 base pairs (E) TYPE: Nucleic acid (F) No. OF HEBRAS: One (G) TOPOLOGY: Linear (v) TYPE OF MOLECULE: Other nucleic acid (A) DESCRIPTION: / desc = "primer of the DNA oligonucleotide chain " (xi) DESCRIPTION OF THE SEQUENCE: IDENTIFICATION OF THE SEQUENCE No: 7: CATACATACA AGCTGTCTTC TGTGGTGTAC CA 32 (2) INFORMATION FOR THE IDENTIFICATION OF SEQUENCE No. 8: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 33 base pairs (B) TYPE: Nucleic acid (C) No. OF HEBRAS: One (D) TOPOLOGY: Linear (iii) TYPE OF MOLECULE: Other nucleic acid (A) DESCRIPTION: / desc = "primer of the DNA oligonucleotide base" (xi) DESCRIPTION OF THE SEQUENCE: IDENTIFICATION OF SEQUENCE No. 8: CGAATAAGAA AGACGTCACT GTTCAGGAGT TGG 33 (2) INFORMATION FOR THE IDENTIFICATION OF THE SEQUENCE No, (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 33 base pairs (B) TYPE: Nucleic acid (C) No. OF HEBRAS: A (D) TOPOLOGY: Linear (iv) TYPE OF MOLECULE: Other nucleic acid (A) DESCRIPTION: / desc = "oligonucleotide DNA series primer" (xi) DESCRIPTION OF THE SEQUENCE: IDENTIFICATION OF SEQUENCE No. 9: CCAACTCCTG AACAGTGACG TCTTTCTTAT TCG 33 (2) INFORMATION FOR THE IDENTIFICATION OF SEQUENCE No. 10 (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 32 base pairs (B) TYPE: Nucleic acid (C) No. OF HEBRAS: One (D) TOPOLOGY : Linear (vi) TYPE OF MOLECULE: Other nucleic acid (E) DESCRIPTION: / desc = "DNA oligonucleotide chain primer" (Xi) DESCRIPTION OF THE SEQUENCE: IDENTIFICATION OF THE SEQUENCE No: 10: GAGATAATAA AGTTATTAAC TCAGAAAACA TG 32 (2) INFORMATION FOR THE IDENTIFICATION OF SEQUENCE No. 11: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 32 base pairs (F) TYPE: Nucleic acid (G) No. OF HEBRAS: One (H) TOPOLOGY: Linear (v) TYPE OF MOLECULE: Other nucleic acid (E) DESCRIPTION: / desc = "primer of the DNA oligonucleotide base" (xi) DESCRIPTION OF THE SEQUENCE: IDENTIFICATION OF SEQUENCE No. 11: CATGTTTTCT GAGTTAATAA CTTTATTATC TC 32 (2) INFORMATION FOR THE IDENTIFICATION OF SEQUENCE No. 12 (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 49 base pairs (B) TYPE: Nucleic acid (C) No. OF HEBRAS: A (D) TOPOLOGY: Linear (ii) TYPE OF MOLECULE: Other nucleic acid (I) DESCRIPTION: / desc = "DNA oligonucleotide chain primer" (xi) DESCRIPTION OF THE SEQUENCE: IDENTIFICATION OF THE SEQUENCE No: 12: CGCGGATCCG CGCGGCACCA GGCCGCTGTT ATCCGGAAAA TGCTCT 9 (2) INFORMATION FOR THE IDENTIFICATION OF SEQUENCE No. 13: (i) CHARACTERISTICS OF THE SEQUENCE: (A) - LENGTH: 77 base pairs (B) TYPE: Nucleic acid (C) No. OF HEBRAS: One (D) ) TOPOLOGY: Linear (ii) TYPE OF MOLECULE: Other nucleic acid (F) DESCRIPTION: / desc = "primer of the DNA oligonucleotide base" (xi) DESCRIPTION OF SEQUENCE: IDENTIFICATION OF SEQUENCE No. 13: CCGGATAACA GCGCGCGTCA GGCTAACGAA CTCCCAGGCG CCCCGTCACA GGAAGAACGC CCGCAGGTCC AACTGCA 77 (2) INFORMATION FOR THE IDENTIFICATION OF SEQUENCE No. 14: (i) CHARACTERISTICS OF THE SEQUENCE:. (A) LENGTH: 69 base pairs (B) TYPE: Nucleic acid (C) 'No. OF HEBRAS: One (D) TOPOLOGY: Linear (ii) TYPE OF MOLECULE: Other nucleic acid (A) DESCRIPTION: / desc = "primer" of the DNA oligonucleotide sequence " (xi) DESCRIPTION OF THE SEQUENCE: IDENTIFICATION OF SEQUENCE No. 14: GTTGGACCTG CGGGCGTTCTTCCTGTGACG GGGCGCCTGG CAGTTCGTTA G CGCTGTTAT 69 (2) INFORMATION FOR THE IDENTIFICATION OF SEQUENCE No. 15: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 18 amino acids (B) TYPE: Amino Acid (C) No. OF HEBRAS: One (D) TOPOLOGY: Linear (ii) TYPE OF MOLECULE: Peptide (xi) DESCRIPTION OF THE SEQUENCE: IDENTIFICATION OF THE SEQUENCE No: 15: Be Ala Arg Gln Ala Asn Glu Leu Pro Gly Ala Pro Ser Gln Glu Glu Arg Ala 1 5 10 15 (2) INFORMATION FOR THE IDENTIFICATION OF SEQUENCE No. 16: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 18 amino acids (B) TYPE: Amino Acid (C) No. OF HEBRAS: One (D) TOPOLOGY: Linear (ii) TYPE OF MOLECULE: Peptide (xi) DESCRIPTION OF THE SEQUENCE: IDENTIFICATION OF THE SEQUENCE No. 16: Be Ala Arg Gln Ala Asn Glu Leu Pro Gly Ala Pro Ser Gln Glu Glu Arg Pro 1 5 10 15 (2) INFORMATION FOR THE IDENTIFICATION OF SEQUENCE No. 17: (i) CHARACTERISTICS OF THE SEQUENCE: (A> LENGTH: 84 base pairs (B) TYPE: Nucleic acid (C) No. OF HEBRAS: One (D) ) TOPOLOGY: Linear (vii) TYPE OF MOLECULE: Other nucleic acid (J) DESCRIPTION: / desc = "primer of the DNA oligonucleotide chain" (xi) DESCRIPTION OF THE SEQUENCE: IDENTIFICATION OF THE SEQUENCE No: 17: GCGGATCCCG GTCCGCGTCA GGCTAACGAA CTGCCAGGAG CTCCGTCTCA GGAAGAGCGT GCACCTACTT CAAGTTCTAC AAAG 84 (2) INFORMATION FOR THE IDENTIFICATION OF THE SEQUENCE No, 18: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 38 base pairs (B) 'TYPE: Nucleic acid (C) No. OF HEBRAS: One (D) ) TOPOLOGY: Linear (vi) TYPE OF MOLECULE: Other Nucleic Acid (A) DESCRIPTION: / desc = "primer of the DNA oligonucleotide base" (xi) DESCRIPTION OF THE SEQUENCE: IDENTIFICATION OF SEQUENCE No. 18: CCGAATTCGC TAGCTTATCAAGTTAGTGTT GAGATGAT 38 (2) INFORMATION FOR THE IDENTIFICATION OF SEQUENCE No. 19: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 11 amino acids (G) TYPE: Amino Acid (H) No. OF HEBRAS: One (I) TOPOLOGY: Linear (ii) TYPE OF MOLECULE: Peptide (xi) DESCRIPTION OF THE SEQUENCE: IDENTIFICATION OF SECUE No .: 19: Pro Wing Being Gly Gly Gly Wing Gly Wing Gly Pro 1 5 10 (2) INFORMATION FOR THE IDENTIFICATION OF SEQUENCE No. 20: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 17 amino acids (B) TYPE: Amino acids (C) No. OF HEBRAS: One (D) TOPOLOGY: Linear (ii) TYPE OF MOLECULE: Peptide (xi) DESCRIPTION OF THE SEQUENCE: IDENTIFICATION OF SEQUENCE No: 20: Gly Pro Arg Gln Ser Asn Glu Thr Pro Gly Ser Pro Ser Gln Glu Glu Arg 1 5 10 15 (2) INFORMATION FOR THE IDENTIFICATION OF SEQUENCE No. 21 (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 17 amino acids (B) TYPE: Amino acids (C) No. OF HEBRAS: One (D) TOPOLOGY: Linear ( ii) TYPE OF MOLECULE: Peptide (xi) DESCRIPTION OF THE SEQUENCE: IDENTIFICATION D SEQUENCE No. 21: Gly Pro Arg Gln Wing Lys Thr Leu Pro Gly Wing Pro Ser Gln Thr Thr Arg 5 10 15 (2) INFORMATION FOR THE IDENTIFICATION OF SEQUENCE No. 22: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 17 amino acids (E) TYPE: Amino acids (F) No. OF HEBRAS: One (G) TOPOLOGY: Linear (ii) TYPE OF MOLECULE: Peptide (xi) DESCRIPTION OF THE SEQUENCE: IDENTIFICATION OF SEQUENCE No: 22: Gly Pro Thr Glu Wing Asp Glu Leu Pro Gly Wing Pro Ser Glu Glu Glu Thr 1 5 10 15 (2) INFORMATION FOR THE IDENTIFICATION OF SEQUENCE No. 23: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 17 amino acids (B) TYPE: Amino Acid (C) No. OF HEBRAS: One (D) TOPOLOGY: Linear (ii) TYPE OF MOLECULE: Peptide (xi) DESCRIPTION OF THE SEQUENCE: IDENTIFICATION OF SEQUENCE No. 23: Gly Pro Arg Gln Wing Asn Glu Leu Pro Gly Wing Pro Ser Gln Glu Glu Arg 1 5 10 15

Claims (32)

1. A method for inactivating the target cells in the presence of T cells by contacting a superantigen (SAG) with the two types of cells in the presence of an immunomodulator, characterized in that at least one superantigen and one immunomodulator are in the form of a conjugate between an independent superantigen (Sag) and a portion that looks for the conjugate in the target cells.
2. The method according to claim 1, characterized in that: a. the superantigen and the immunomodulator are used in the form of a triple conjugate comprising a superantigen (Sag), a portion that searches (T) for the target cells and an immunomodulator (IM) (conjugate-Sag T, IM); b. the superantigen (SAG) is used in the form of a double conjugate between a superantigen (Sag) and a portion that searches (T) for target cells in combination with a double conjugate between an immunomodulator (IM) and a searching portion (T '). ) to target cells (conjugate-Sag T, + conjugate-IM T '); c. The superantigen (SAG) is used in the form of a double conjugate between a superantigen (Sag) and a portion that searches (T) for the target cells and the innornodulator (IM) that is used independently, that is, it is not conjugated with a portion that searches the target cells (conjugate-Sag T, + IM); d. the superantigen (SAG) is used independently (Sag) and the immunomodulator is used in conjugated form, that is, a double conjugate between the immunomodulator and a target-seeking portion (Sag + conjugate-IM T,); and e. The superantigen (SAG) and the immunomodulator are used in the form of a double conjugate between a superantigen (Sag) and an immuno modulator (IM) (conjugate-IM, Sag).
3. The method according to claim 2, characterized in that alternative a is used.
4. The method according to claim 2, characterized in that the alternative b is used, with the possibility that the portion that looks for the conjugated immunomodulator may differ from the portion searching for the conjugated superantigen.
5. The method according to claim 2, characterized in that alternative c is used.
6. The method according to claim 2, characterized in that the alternative e y is used in that IM and T is common for, for example, a cytokine receptor, such as 11-2, to go to the conjugated cells that transport the receptor.
7. The method according to any of claims 1-6, characterized in that the cells are inactivated in vivo in an individual suffering from disease associated with the target cells, for example, a cancer.
8. The method according to any of claims 1-7, characterized in that the portion that searches the target is an antibody, preferably an antigen binding fragment thereof, such as a Fab or a Fab2 fragment or a single chain antibody .
9. The method according to any of claims 1-7, characterized in that the Sag superantigen is modified, for example, by a mutation, a. so that it has a decreased ability to bind MHC class II antigen compared to the wild type superantigen; b. so that it has a decreased serosensitivity in human serum compared to the corresponding wild-type superantigen; c. so that it has a decreased immunogenicity in the human compared to the corresponding wild-type superantigen; d. so you have a chimera between two or more independent superantigens.
10. . The method according to any of claims 1-9 characterized in that the superantigen is modified at a reduced affinity for the MHC class II, for example, by a mutation in a codon encoding an amino acid residue of importance for affinity with the MHC class II.
11. 11. The method according to any of claims 1-10 characterized in that the immunomodulator is selected from: a. cytokines, such as IL-2, or b. chemokines or c. extracellular parts of ligands and receptors agglutinated to the surface of lymphocytes, for example, the extracellular parts of a B7 molecule, such as CD80 and CD86.
12. 12. The method according to any of claims 1-11, characterized in that the immunomodulator is selected from immunomodulators that are capable of potentiating the effects of superantigens in vivo, for example, by counteracting the escape in anergy of activated T cells by superantigens.
13. 13. The method according to any of claims 1-11, characterized in that the immunomodulator is the extracellular part of a B7 ligand, such as CD80 or CD86, with an effector in -direction 3 'of the CD28 / B7 signaling, such as IL-2.
14. 14. The method according to any of claims 11-13, characterized in that the immunomodulator has been modified, for example, by a mutation to show a decreased affinity towards its lymphocyte receptor, compared to that of the natural form.
15. 15. The method according to any of claims 1-13, characterized in that the immunomodulator is IL-2 or the extracellular part of CD80 or forms thereof that have been modified in accordance with claim 14.
16. 16. A conjugated superantigen according to the formula: (T) x (Sag) and (IM) z Formula I characterized in that: a. T is a portion that searches the target, Sag corresponds to an independent superantigen, IM is an immunomodulator which is not a superantigen and T, Sag and IM are linked by organic binders B which may be different or equal within one and the Same conjugated molecule. b. x, y, and z are integers that are typically selected from 0-10, such as 0-5, and represent the number of T, Sag and IM portions, respectively, in a given conjugate molecule, with the proviso that and > 0 and also one or both of x, z > 0;
17. 17. The conjugated superantigen according to claim 16, characterized in that it is a fusion protein where all x and Y and z are integers 1-3, preferably 1--2, and the typical relationships between x, y, z being selected between x = y = z; x = y = 0.5z; x = 0.5y = 0.5z; and x = 0.5y = z.
18. 18. The conjugated superantigen according to claim 16, characterized in that it is a fusion protein that is expressed as a double-stranded product.
19. 19. The fusion protein according to claim 18 characterized in that the portion of the Sag superantigen is fused C-terminally to the target seeking portion T 'and the immunomodulator IM is fused C-terminally to the portion seeking the objective T ''.
20. . The fusion protein according to claim 19 characterized in that T 'and T "are defined in claim 8 and / or SAG is as defined in any of claims 9-10 and / or IM is as defined in any of of claims 11-15.
21. . The fusion protein according to claim 20 characterized in that SAG is staphylococcal enterotoxin A (SEA), T 'is the -CH1 domain of the Fab C215 fragment, T' 'is the light chain of the C215 antibody and IM is interleukin -2.
22. . The fusion protein according to claims 1-21 characterized in that SAG is fused to T 'by a flexible hydrophilic amino acid B' linker of 3-11 amino acid residues and IM is fused to T '' by the amino acid Q linker with neutral or positive charge with 10-20 amino acid residues.
23. The fusion protein according to claim 22, characterized in that B 'is selected from the group consisting of: Gly-Gly-Pro and Pro-Ala-Ser-Gly-Gly-Gly-Gly-Ala-Gly-Gly-Pro (SEQ ID NO: 19) and Q is selected from the group consisting of Gly-Pro-Arg-Gln -Ala-Asn-Glu-Leu-Pro-Gly-Ala-Pro-Ser-Gln-Glu-Glu-Arg (SEQ ID No. 23), Gly-Pro-Arg-Gln-Ser-Asn-Glu-Tr- Pro-Gly-Ser-Pro-Ser-Gln-Glu-Glu-Arg (SEQ ID No. 20), Gly-Pro-Arg-Gln-Ala-Lis-Tr-Leu-Pro-Gly-Ala-Pro-Ser -Gln-Tr-Tr-Arg (SEQ ID No. 21) and Gly-Pro-Tr-Glu-Ala-Asp-Glu-Leu-Pro-Gly-Ala-Pro-Ser-Glu-Glu-Glu-Tr ( SEQ ID No. 22).
24. The conjugated superantigen according to claim 16, characterized in that it is a fusion protein, the target seeking portion is absent (x = 0) and, z are integers 0-3, preferably 1-2 and the relationships preferred for x, and are selected from among x = y; x = 0.5y; 0.5y = x; x = l / 3y and l / 3x = y.
25. The conjugated superantigen according to claim 16, characterized in that it has the formula: (Sag) and (IM) z Formula II which y = z = 1.
26. 26. The conjugated superantigen according to claims 16, 17, 24 or 25, characterized in that the target seeking portion is as defined in claim 8 and / or the portion of the superantigen is as defined in any of claims 9- 10 and / or the immunomodulator portion is as defined in any of claims 11-15.
27. 27. A sought immunomodulator, characterized in that it is a conjugate between a targeting portion (T '' ') and an immunomodulator (IM' '') that is not a superantigen which has been modified, for example, by a mutation, to have a decreased affinity towards its receptor or to have a diminished proportion of internalization when it agglutinates to its lymphocyte receptor (compared to the corresponding natural form), the aforementioned conjugate fulfilling the formula: (T "') x (Sag'") and (I - / ") z Formula V where: a. T '' 'is a portion that seeks the target, Sag 'corresponds to an' independent superantigen, IM '' 'is the modified immunomodulator, Sag and IM are joined by organic binders B' '' which may be different or equal within one and the same conjugate molecule, b. x, y, z are integers that are typically selected from 0-10, such as 0-5, and represent the number of portions T '' ', Sag' '' and IM '' ', respectively, in a conjugate molecule given, with the proviso that z > 0 and also that one or both x, y > 0
28. The conjugated immunomoduladsr searched. according to claim 27, characterized in that it is a fusion protein where all x, y, z are integers 1-3, preferably 1-2 and the typical relationships between x, y, z are selected from among x = y = z; x = y = 0.5z; x = 0.5y = 0.5z; and x = 0.5y = z.
29. . The immunomodulator sought, according to claim 27, characterized in that it is a fusion protein, the proportion of the superantigen is absent (y = 0) y. x. z are integers 1-3, preferably 1-2, the preferred relationships between x, y are selected from x = y; x = 0 5y; 0 5x = y; x = l / 3y; and l / 3x = y.
30. 30. The immunomodulator sought in accordance with claim 29, characterized in that it complies with the formula (T '' ') and (IM' ") z in which y = z = 1.
31. 31. A DNA molecule characterized in that it encodes a superantigen and an immunomodulator, such as IL-2, which is not a superantigen.
32. 32. The DNA molecule according to claim 31, characterized in that it is in the form of a biscistronic structure in which: a. a first cistron contains a sequence I encoding a polypeptide I comprising a non-conjugated superantigen (Sag) which is possibly modified as defined in claims 9-10, and b. the other cistron contains a sequence II that codes for a polypeptide II comprising the immunomodulator that is possibly modified as defined in claims 11-15. . The DNA molecule according to claim 32, characterized in that either or both of sequences I and II are fused in a respective sequence encoding at least a portion of an antibody such that polypeptides I and II can associate and forming a triple fusion comprising an independent superantigen, an immunomodulator and an antibody. . The DNA molecule according to claim 31, characterized in that the superantigen is a non-conjugated superantigen and in which the sequence encoding the superantigen is fused to the sequence encoding the immunomodulator possibly by means of a sequence encoding a ligand Oligopeptides. SUMMARY OF THE INVENTION A method for inactivating target cells in the presence of T cells by contacting the two cell types with a superantigen (SAG) in the presence of an immunomodulator, characterized in that at least one superantigen and the immunomodulator are in the form of a conjugate between the "independent" superantigen (Sag) and a portion that targets the conjugate for the target cells. A conjugated superantigen that complies with formula (I): (T) x (Sag) and (IM) z; a) T is a portion that searches for the target, Sag corresponds to an independent superantigen, MI is an immunomodulator which is not a superantigen and .T, Sag and IM are linked to each other by organic binders B; b) x, y, and z are integers that are typically selected from 0-10 and represent the number of T, Sag and IM portions, respectively, in a given conjugate molecule, with the proviso that y is>. 0 and either also x or z > 0. The conjugated superantigen is preferably a triple fusion molecule. An objective immunomodulator, characterized in that it is a conjugate between a targeting portion (T "'') and a modified immunomodulator (IM" ''). The conjugate fulfills a formula analogous to formula (I) except for the indispensable presence of the modified immunomodulator. A portion of the superantigen may be present. A DNA molecule that codes for a superantigen and an immunomodulator.