A method for the treatment of a disease in a mammal by administering a therapeutically effective amount of a conjugate comprising a biospecific affinity counterpart and a peptide, wherein the peptide contains an amino acid sequence that is derived from staphylococcal enterotoxin A, binds to a V.beta. of a T cell receptor, and has a D227A mutation so that the peptide has a modified ability to bind to MHC class II antigens.

THE OBJECTIVES OF THE INVENTION

A first objective of the invention is to improve previously known superantigen-antibody conjugates with respect to general immune stimulation versus directed cytotoxicity. Stimulation results in activated T-lymphocytes and is dependent on the ability of the superantigen to bind to both the T cell receptor and an MHC class II antigen.

A second objective of the invention is to provide conjugates between biospecific affinity counterparts (e.g. antibodies) and superantigens with a modified affinity for MHC class II antigens. This has now been shown to improve the selectivity for superantigen antibody dependent cell cytolysis (SADCC) of cells exposing the antigen (against which the antibody/biospecific affinity counterpart of the conjugate is directed) over other cells exposing MHC class II antigens.

A third objective of the invention is to provide conjugates that can be used as the active principle in the treatment of mammals suffering from cancers, autoimmune diseases, parasitic infestations, viral infections or other diseases associated with cells that on their surface express structures that are specific for respective disease.

The Invention

The main aspect of the invention is a conjugate comprising a. a biospecific affinity counterpart that is directed towards a structure to which one intends to bind to the conjugate, b. a peptide that i. is derived from a superantigen, ii. has the ability to bind to the V.beta. chain of the T cell receptor, and iii. has a modified ability to bind to MHC class II antigens compared to the superantigen from which the peptide is derived (wild-type of superantigen=SA(wt)).

The peptide and the affinity counterpart are covalently linked to each other via a bridge (B).

The preferred conjugates have the ability to activate and direct T-lymphocytes to selective lysis of cells that on their surface expose the structure against which the affinity counterpart is directed. This means that the conjugates shall cause cytolysis in an SADCC mediated method (Superantigen Antibody Dependent Cellular Cytotoxicity). See the experimental part below and our previous publications concerning conjugates between superantigens and antibodies (e.g. Dohlsten et al., WO 9201470).

The inventive conjugates have a structure that is analogous to the superantigen-antibody conjugates described in the prior art (Dohlsten et al., WO 9201470 which hereby is incorporated by reference), i.e. the conjugates complies with the formula: T--B--SA(m) where T represents the biospecific affinity counterpart, SA(m) is the modified superantigen (the above-mentioned peptide), and B is a covalent bridge linking T and SA(m) together.

T can in principle be any structure that binds via biospecific affinity. In most important cases, T is capable of binding to a cell surface structure, preferably a disease specific structure as given above. The structure against which T is directed is usually different from (a) the V.beta. chain epitope to which the superantigen derived peptide (SA(m)) binds and (b) the MHC class II antigen epitope to which the unmodified superantigen binds. The biospecific affinity counterpart T may primarily be selected among interleukins (e.g. interleukin-2), hormones, antibodies and antigen binding fragments of antibodies, growth factors etc. See for instance Woodworth, Preclinical and Clinical Development of Cytokine Toxins presented at the conference "Molecular Approaches to cancer Immunotherapy", Ashville, N.C., Nov. 7 11, 1993. Polypeptides binding to the constant domains of immunoglobulins (e.g. Proteins A and G and L), lectins, streptavidin, biotin etc were at the priority date considered to be of minor importance.

At the priority date, it was preferred that T was an antibody or an antigen binding fragment of an antibody (including Fab, F(ab).sub.2, Fv, single chain antibody etc), with particular emphasis of an antibody active fragment (such as Fab) of antibodies directed against the so called C242 epitope (Lindholm et al., WO 9301303) or against other cancer specific epitopes.

In case T is an antibody it is primarily monoclonal or a mixture of a defined number of monoclonals (e.g. 2, 3, 4, 5 or more). T may be a polyclonal antibody, in case the use is non-therapeutical.

It is not imperative for T to have a polypeptide structure. The modified superantigen SA(m) is primarily a mutated superantigen but may potentially also be a chemically modified superantigen, including fragments of superantigens retaining the ability to bind to the V.beta. chain of the T cell receptor.

The expression "mutated superantigen" means that the native ability of the superantigen to bind to MHC class II antigens has been modified on the genomic level by replacing, inserting or removing one or more amino acids in the native superantigen.

Superantigen fragments obtained by mutations removing parts of the full amino acid sequence and fragments obtained by enzymatic or chemical cleavage of superantigens may be used equivalently in chemical conjugates of the invention.

The modified superantigen SA(m) may comprise one or more amino acid sequences that are derived from different superantigens and that may have been mutated, for instance combinations of the preferred superantigens mentioned below.

The modified superantigen SA(m) as such may exhibit a decreased immunogenicity and toxicity compared to the native superantigen.

Other groups/substances that are capable of cross reacting with the V.beta.-chain of the T cell receptor may potentially also be employed equivalently with the mutated superantigen (SA(m)) as given above. Such groups/substances may be of non-polypeptide structure.

At the end of the priority year the most interesting product candidates of the invention comprised mutated forms of superantigens having multiple MHC class II binding sites and/or the ability to coordinate Zn.sup.2+, for instance SEA, SED, SEE and SEH.

T as well as SA(m) may be prepared by recombinant techniques.

The bridge B may be selected as previously described (Dohlsten et al., WO 9201470), i.e. it shall preferably be hydrophilic and exhibit one or more structure(s) selected among amide, thioether, ether, disulfide etc. In case the bridge have unsubstituted unbroken hydrocarbon chains they preferably lack aromatic rings, such as phenyl. The most important bridges are those obtained by recombinant techniques, i.e. when the conjugation takes places on the genomic level. In such cases oligopeptide bridges containing hydrophilic amino acid residues, such as Gln, Ser, Gly, Glu and Arg, are preferred. Pro and His may also be included. During the priority year it has been decided that the preferred bridge is a peptide comprising three amino acid residues (GlyGlyPro).

The inventive conjugate may comprise one or more modified superantigen(s) per biospecific affinity counterpart and vice versa. This means that T in the formula above may contain one or more modified superantigens in addition to the biospecific counterpart. In analogy SA(m) may contain one or more biospecific affinity counterpart(s) T. The affinity counterpart T and SA(m) may also comprise other structures. The number of modified superantigens per affinity counterpart is preferably one or two. The synthesis of the novel inventive conjugates may be carried out in principle according to two main routes: 1. by recombinant techniques and 2. chemical linking of T to SA(m). The methods are well recognized for the ordinary skilled worker in the field and comprise a large number of variants. It follows that the invention primarily concerns artificial conjugates, i.e. conjugates that are not found in nature.

Chemical linking of a modified superantigen to the biospecific affinity counterpart T often utilizes functional groups (e.g. primary amino groups or carboxy groups) that are present at many positions in each compound. It follows that the final product will contain a mixture of conjugate molecules differing with respect to the position at which linking has taken place.

For recombinant conjugates (fusion proteins) the obtained conjugate substance will be uniform with respect to the linking position. Either the amino terminal of the modified superantigen is linked to the carboxy terminal of the biospecific affinity counterpart or vice versa. For antibodies, such as intact antibodies and antigen binding fragments (Fab, Fv etc), either the light or the heavy chain may be utilized for such fusions. At present time recombinant conjugates are preferred, with preference for Fab fragments and linking of the amino terminal of the modified superantigen to the first constant domain of the heavy antibody chain (CH1), without exclusion of the analogous linking to the light chain or to the VH and VL domain that also may give quite good results.

There are two different methods for obtaining large amounts of superantigens (including modified and fused forms) in E. coli: intracellular production or secretion. The latter method is preferred for the inventive conjugates because it offers purification of correctly folded protein from the periplasma and from the culture medium. Intracellular production results in a complicated purification procedure and often needs refolding in vitro of the protein (in order for the protein to obtain the correct tertiary structure). The above does not exclude that it is possible to produce active conjugates also in other host cells, e.g. eukaryotic cells, such as yeast or mammalian cells.

The production of mutated superantigens and selection of mutants having a modified ability to bind (affinity) to MHC class II antigens may be carried out according to known techniques (se e.g. Kappler et al., J. Exp. Med. 165 (1992) 387 396). See also our experimental part.

The ability of the conjugate to bind to the T cell receptor V.beta. chain, to the target structure and to cause lysis of the target cell depends on i.a. the peptide (SA(m)) that is derived from a superantigen, the biospecific affinity counterpart (T) and the structure and length of the bridge (B). A person ordinary skilled in the art is able to optimize the inventive conjugates with respect to the binding ability and the ability to cause lysis by studying the relationship between effect and structure with the aid of those models that have been disclosed in connection with previously known superantigen antibody conjugates (see the above-referred publications). See also the experimental part below.

By modified ability to bind MHC class II antigens is primarily intended that the ratio IC.sub.50(SA(wt)):IC.sub.50(SA(m)) is <0.9 (90%), such as <0.5 (<50%) and possibly also <0.01 (<1%). In the alternative the modified binding ability of the inventive conjugates can be measured as the ratio of the dissociation constants K.sub.d(SA(wt)):K.sub.d(SA(m)) with K.sub.d measured in nM and with the same limits as for the ratio IC.sub.50(SA(wt)):IC.sub.50(SA(m)). For the determination of IC.sub.50(SA(wt), IC.sub.50(SA(m)), K.sub.d(SA(m)) and K.sub.d(SA(m)) see the experimental part below.

It is previously known that certain superantigens may have two or more sites that bind to MHC class II antigen (Fraser et al., In: Superantigens: A pathogens view on the immune system. Eds. Huber & Palmer, Current Communications in Cell Molecular Biology 7 (1993) 7 29). For this type of superantigens the binding ability shall be modified at least one of the binding sites, e.g. as a reduction of the above-mentioned size. Possibly it may suffice with a superantigen modification that create a changed difference in affinity for two MHC class II binding sites, tentatively >10% and preferably by reducing the affinity of at least one site.

Superantigens bind to TCR V.beta. chains of different subgroups with varying affinities. In the inventive fusion proteins/conjugates, the superantigen employed may have been modified so as to show an altered subgroup specificity or an altered affinity to one or more members of the subgroup. There are strong reasons to believe that a parabolic relationship exists between the affinity for TCR V.beta. and stimulation via TCR, i.e. a moderate affinity will give the maximal stimulation. Accordingly an appropriate affinity of a modified superantigen for TCR V.beta. may be at hand as soon as the fusion protein/conjugate comprising the modified superantigen is able to significantly stimulate a resting T cell population representing essentially the distribution of all human V.beta. subgroups to proliferate. The T cell population may be pooled T cells from randomly selected human individuals. By significantly is meant that the stimulation is possible to measure. The results presented in Table II (right column) in the experimental part indicate that the ability to cause SADCC of the inventive conjugates/fusion proteins often is essentially the same as for the fusion comprising the wild-type superantigen.

Main Use of the Conjugates/Fusion Proteins of the Invention.

The conjugates according to the invention are primarily intended for the treatment of the same diseases as the conjugates between normal superantigens and antibodies. See the above-mentioned publications. Thus the inventive conjugates may be administered either as the main therapy or as adjuvant therapy in connection with surgery or other drugs.

The pharmaceutical composition of the invention comprises formulations that as such are known within the field but now containing our novel conjugate. Thus the compositions may be in the form of a lyophilized particulate material, a sterile or aseptically produced solution, a tablet, an ampoule etc. Vehicles such as water (preferably buffered to a physiologically pH value by for instance PBS) or other inert solid or liquid material may be present. In general terms the compositions are prepared by the conjugate being mixed with, dissolved in, bound to, or otherwise combined with one or more water-soluble or water-insoluble aqueous or non-aqueous vehicles, if necessary together with suitable additives and adjuvants. It is imperative that the vehicles and conditions shall not adversely affect the activity of the conjugate. Water as such is comprised within the expression vehicles.

Normally the conjugates will be sold and administered in predispensed dosages, each one containing an effective amount of the conjugate that, based on the result now presented, is believed to be within the range of 10 .mu.g 50 mg. The exact dosage varies from case to case and depends on the patient's weight and age, administration route, type of disease, antibody, superantigen, linkage (--B--) et.

The administration routes are those commonly known within the field, i.e. a target cell lysing effective amount or a therapeutically effective amount of a conjugate according to the invention is contacted with the target cells. For the indications specified above this mostly means parenteral administration, such as injection or infusion (subcutaneously, intravenously, intra-arterial, intramuscularly) to a mammal, such as a human being. The conjugate may be administered locally or systemically.

By "target cell lysing effective amount" is contemplated that the amount is effective in activating and directing T-lymphocytes to destroy the target cell.

At the end of the priority year it had been decided that the preferred administration route for conjugates/fusion proteins comprising unmodified superantigens is 3 hours' intravenous infusion per day combined with a fever-reducing agent (paracetamol). The administration is to be repeated during 4 days and stopped before dsecondary antibodies are raised against the fusion protein/conjugate in the patient. This dosage schedule is likely to be applicabple also to the present inventive conjugates/fusion proteins.

Alternative Fields of Use.

The inventive conjugates can also be employed to quantitatively or qualitatively detect the structure against which the target-seeking group (T) is directed. In general these methods are well-known to people in the field. Thus, the modified superantigen may function as a marker group within immunoassays including immunohistochemistry meaning that the marker group in turn is detected by for instance an antibody that is directed towards the peptide (SA(m)) and labelled with an enzyme, isotope, fluorophor or some other marker group known per se. Another immunoassay method is to detect in a cell population cells that on their surface express a structure capable of binding to the target-seeking group (T). This use means that a sample from the cell population is incubated with T-lymphocytes together with the present inventive conjugate as in an SADCC assay. In case the incubation leads to cell lysis this is an indication that the population contains cells that on their surface express the structure.
 

Claim 1 of 2 Claims

1. A method for the treatment of a disease condition in a mammal, which condition means the presence of specific cells that are associated with the condition by the expression of a disease specific cell surface structure, wherein one administers to the mammal a therapeutically effective amount of covalent conjugate that is able to activate T lymphocytes to lyse cells that carry the disease specific cell surface structure and comprises: a. a biospecific affinity counterpart that is capable of binding to said surface structure, and b. a peptide that i. contains an amino acid sequence that is derived from staphylococcal enterotoxin A, wherein said peptide has the ability to bind to a V.beta. of a T cell receptor, and ii. has been mutated in that amino acid substitution D227A has been made in staphylococcal enterotoxin A to show a modified ability to bind to MHC class II antigens.

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