Modified/chimeric superantigens and their use

Details Modified/chimeric superantigens and their use Modified/chimeric superantigens and their use

1996-08-12

2003-02-04

Ungar, Susan (Department: 1642)

Drug, bio-affecting and body treating compositions

Conjugate or complex of monoclonal or polyclonal antibody,...

C424S182100, C424S184100, C424S185100, C424S190100, C424S192100, C424S194100

Reexamination Certificate

active

06514498

ABSTRACT:

This application claims priority from Swedish Patent Application No 9601245-5, which was filed Mar. 29, 1996, and is incorporated herein by reference.
FIELD OF THE INVENTION
The present invention relates to functionally active modified superantigens which are wild-type superantigens (SA I) in which one or more amino acid residues have been substituted while maintaining superantigen function. In case one or more of the substituting residues (or a conserved amino acid residue thereof) occur in the corresponding positions in another wild-type superantigen (SA II), the modified superantigen is called a chimera. Chimeric superantigens thus will contain part sequences/regions deriving from at least two different wild-type superantigens.
By the term “corresponding” is meant that residues, part sequences and regions replacing each other have functionally the same position in superantigens I and II so that substitution will lead to a chimeric form that is able to function as a superantigen.
The terminology grafted/grafting/graft is used in connection with parts of the full sequence of superantigen II that have replaced corresponding parts of superantigen I, even if only one single amino acid has been replaced.
Modified/chimeric superantigens also encompass functional superantigens modified in other ways, for instance conjugated to a target-seeking moiety, including also fused forms when the moiety is a polypeptide/protein. See below.
Superantigens
According to the very first definition (around 1988-1993), superantigens are bacterial or viral proteins capable of binding to MHC class II antigens without prior intracellular processing and activate T cells by binding to the &bgr;-chain variable region (V &bgr;) of the T cell receptor (TCR). The binding leads to a V&bgr; family restricted activation of a relatively large proportion/subset of T cells and lysis of MHC Class II expressing cells (superantigen dependent cell cytolysis=SDCC).
Well known wild-type superantigens according to the definition above are the staphylococcal enterotoxins (SEA, SEB, SEC1, SEC2, SED, SEE and SEH). Further examples are Toxic Shock Syndrome Toxin 1 (TSST-1, also of staphylococcal origin), Exfoliating Toxins (EXft), Streptococcal Pyrogenic Exotoxin A, B and C (SPE A, B and C), Mouse Mammary Tumor Virus proteins (MMTV), Streptococcal M proteins, Clostridial Perfringens Enterotoxin (CPET), mycoplasma arthritis superantigens etc. For a review of superantigens and their properties see Kotzin et al 1993.
During the early nineties it was discovered that activation and subsequent cell lysis could occur in a MHC class II independent manner in case the superantigen was conjugated with a target-seeking moiety capable of binding to a cell surface structure (Dohlsten et al WO9201470 and Abrahmsén et al WO9601650). Upon incubation of target cells (carrying the target structure for the target-seeking moiety) and effector cells (T cells) with the conjugates, the target cells become lysed (superantigen antibody dependent cell cytolysis=SADCC) without any requirement for class II expression. Accordingly the superantigen concept of today and used in the context of the present invention, if not otherwise specified, encompasses any compound (preferably of polypeptide structure) that is capable of binding to a cell surface structure (target structure) and to one or more polymorphic TCR chain, in particular the V&bgr; chain, thereby activating a subset of T cells expressing the specific TCR chain involved in the binding. The T cells then become cytotoxic for cells carrying the surface structure (target structure, target cells). Normally the activated subset of T cells constitutes about 1-20% of the total amount of T cells of an individual.
Background Art—Structural and Functional Studies Utilizing Mutated and Chimeric Superantigens
Chimeric superantigens including point mutated forms have previously been described (Kappler et al WO 9314364, Kappler et al 1992; Grossman et al 1991; Hufnagle et al 1991; Hartwig et al 1993; Fraser et al 1993; Mollick et al 1993; Erwin et al 1992; and Hudson et al 1993). Mollick et al and Hudson et al show from studies of chimeras that the V&bgr; specificity of SEA and SEE resides in certain amino acid sequences present in the carboxy terminal region (i.e. amino acid residues 200, 206 and 207). In addition to the V&bgr; specificity, mainly depending on this region, Mollik et al also were able to show that for complete reconstitution of SEE like activity of SEA containing SEE grafts towards V&bgr;8, a fragment containing the N-terminal 70 amino acid residues from SEE was needed. This fragment contains parts of the SEE-like MHC class II &agr; chain binding site and chimeric SEA/SEE molecules containing this part from SEE, inhibited binding of SEA to MHC class II DR1 in a SEE-like manner.
Recently SEE-SEA chimers involving an exchange of regions involved in binding to TCRV&bgr; have been described (Lamphaer et al., J. Immunol. 156 (Mar. 15, 1996) 2178-2185). A SEE superantigen Fab antibody fusion protein in which the SEE domains involved in the interaction with T cells have been replaced with the corresponding non-homologous SEA domains has been discussed at ABRF'96: Biomolecular Techniques, Holiday Inn Golden Gateway, San Francisco, Calif. Mar. 30-Apr. 2, 1996 (Björk et al., M45).
Background Art—Therapeutic Use of Superantigens
Non-conjugated superantigens have been suggested for therapy with curative effect presumably being accomplished through a general activation of the immune system (Kalland et al WO9104053; Terman et al WO9110680 and WO9324136; Newall et al 1991).
It has also been suggested to use modified superantigens conjugated to target-seeking moieties (Dohlsten et al WO9201470; Abrahmsén et al WO9601650, both hereby being incorporated by reference). This enabled a broader therapeutic use of T cell activation through V&bgr;. The conjugates studied so far have had a diminished class II affinity, which in turn has lead to a decrease of the severe systemic toxicity normally associated with the wild-type superantigens.
Terman et al (WO9110680; WO9324136) in side-sentences suggested cancer therapy with modified superantigens and superantigen fragments.
Kappler et al (WO9314634) have suggested to use non-conjugated to superantigens mutated to have lost their V&bgr;-binding ability (in the context of vaccines). Abrahmsén et al (WO9601650) have suggested cancer therapy with conjugated superantigens having a modified, preferably decreased, ability to bind to Class II antigens. The modifications encompassed single mutations as well as construction of chimeras between different superantigens.
The Problems that Have Been the Objective to Solve with the Present Invention
The sera of human populations normally contain high titers of antibodies against superantigens. For the staphylococcal superantigens, for instance, the relative titers are TSST-1>SEB>SEC1>SE3>SEC2>SEA>SED>SEE. These relative titers indicate immunogenicity problems and problems with neutralizing antibodies in case SEs are administered parenterally. Based solely on these problems, SEE should be the preferred staphylococcal superantigen. In the context of work with fusion proteins, however, we have found that the ability for T cell MHC class II independent cytotoxicity, superantigen-antibody dependent cell cytotoxicity (SADCC), of SEE conjugates is poor. The anti-SE titers also indicate that there might be advantages in modifying a “high titer” superantigen to be more like a “low titer” superantigen.
The Objectives of the Present Invention
A first objective is to improve the previously known superantigens with respect to lowering their immunogenicity and reaction with neutralizing antibodies.
A second objective is to provide superantigens with less side effects when used as a drug.
A third objective is to provide improved superantigens that can be used as the active principle in the treatment of mammals suffering from cancers, autoimmune diseases, parasitic infestations, viral infections or other diseas

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