The present invention relates to compositions and methods of use, wherein the composition comprises a conjugate of a bacterial superantigen and an antibody moiety. More particularly, the bacterial superantigen has been modified to decrease seroreactivity with retained superantigen activity.

BACKGROUND OF THE INVENTION

This application claims priority to the Swedish Application entitled "A Novel Engineered Superantigen for Human Therapy", filing No. 0102327-4 filed Jun. 28, 2001, which is incorporated herein by reference in its entirety.

1. Field of the Invention

The present invention relates to the field of immunology and proliferative diseases, such as cancer. More particularly, it relates to compositions and methods of use, wherein the compositions comprise superantigens that have been modified to reduce seroreactivity.

2. Related Art

Superantigens (SAg's) constitute a group of bacterial and viral proteins that are extremely efficient in activating a large fraction of the T-cell population. Superantigens bind directly to the major histocompatibility complex (MHC) without being processed. In fact, the superantigens bind unprocessed outside the antigen-binding groove on the MHC class II molecules, thereby avoiding most of the polymorphism in the conventional peptide-binding site. The mechanism of binding depends on the superantigen binding to the T-cell receptor (TCR) in the V.beta. chain, instead of binding to the hypervariable loops of the T-cell receptor (TCR).

Staphylococcal enterotoxins (SEs) are a homologous group of superantigens, with regard to both structure and function (Papageorgiou et al., 2000). They are known to be the major cause of food poisoning and toxic shock syndrome in humans.

A novel SAg-based tumor therapeutic approach has been developed for the adjuvant treatment of solid tumors. It utilizes both main arms of the immune system by incorporating the Fab part of a tumor-specific monoclonal antibody and a T-cell activating SAg in a single recombinant fusion protein. Fab-SAg proteins bound to tumor cells can trigger SAg-activated cytotoxic T-cells to kill the tumor cells directly by superantigen antibody-dependent cell mediated cytotoxicity, SADCC. In addition, activated T-cells produce tumoricidal and pro-inflammatory cytokines counteracting the problems of tumor heterogeneity, and macromolecular uptake, respectively.

Superantigen-based tumor therapeutics have had some success, however, one clinical problem that needs to be addressed is the activation of the systemic immune system. Fusion proteins with wildtype SEA have been investigated in clinical trials of colorectal and pancreatic cancer (Alpaugh et al., 1998). Even though encouraging results were obtained, limitations have been observed. Firstly, the product was very toxic. Secondly, preformed antibodies against the superantigens in the patients made the dosing complex. In addition, the product was immunogenic. Therefore repeated cycles of therapy was only possible in a limited number of patients.

Until the present invention, SAg-based therapies were dose-limiting. The present invention is the first to modify a superantigen resulting in decreased seroreactivity with retained superantigen activity; thus, the present invention is novel and non-obvious.

BRIEF SUMMARY OF THE INVENTION

The foregoing has outlined rather broadly the features and technical advantages of the present invention in order that the detailed description of the invention that follows may be better understood. Additional features and advantages of the invention will be described hereinafter which form the subject of the claims of the invention. It should be appreciated by those skilled in the art that the conception and specific embodiment disclosed may be readily utilised as a basis for modifying or designing other structures for carrying out the same purposes of the present invention. It should also be realised by those skilled in the art that such equivalent constructions do not depart from the spirit and scope of the invention as set forth in the appended claims. The novel features which are believed to be characteristic of the invention, both as to its organisation and method of operation, together with further objects and advantages will be better understood from the following description when considered in connection with the accompanying figures. It is to be expressly understood, however, that each of the figures is provided for the purpose of illustration and description only and is not intended as a definition of the limits of the present invention.

In the present invention, it is provided a conjugate comprising a bacterial superantigen and an antibody moiety, wherein the superantigen is a low titer superantigen comprising regions A to E, which region A is a TCR binding site, and regions B to E determine the binding to MHC class II molecules; and the DNA sequence coding for the superantigen is substituted so that no more than 15 amino acid residues in region A are replaced with different amino acids, such that the substituted superantigen has reduced seroreactivity compared to the superantigen from which it is derived; and wherein the antibody moiety is a full length antibody or any other molecule binding antibody active fragment, which is directed against a cancer-associated cell surface structure. Examples of superantigens include, but are not limited to a staphylococcal enterotoxin (SE), a Streptococcus pyogenes exotoxin (SPE), a Staphylococcus aureus toxic shock-syndrome toxin (TSST-1), a streptococcal mitogenic exotoxin (SME) and a streptococcal superantigen (SSA). In specific embodiments, the staphylococcal enterotoxin is staphylococcal enterotoxin A (SEA) or staphylococcal enterotoxin E (SEE).

In specific embodiments, the amino acid residue positions in region A to be replaced are selected from the group consisting of 20, 21, 24, 27, 173 and 204. It is also contemplated that region C may comprise substitutions in no more than 15 amino acid residues. These substitutions may occur at the amino acid residue positions of 79, 81, 83 and 84. Yet further, region E may comprise substitutions of no more than 15 amino acid residues, in which a substitution may occur at amino acid residue position 227.

In another embodiment of the present invention, it is provided a conjugate comprising a bacterial superantigen and an antibody moiety, wherein the superantigen is a low titer superantigen comprising regions A to E, which region A is a TCR binding site, and regions B to E determine the binding to MHC class II molecules; and the amino acid sequence of the superantigen is substituted so that no more than 15 amino acid residues in region B are replaced with different amino acids, such that the substituted superantigen has reduced seroreactivity compared to the superantigen from which it is derived; and wherein the antibody moiety is a full length antibody or any other molecule binding antibody active fragment, which is directed against a cancer-associated cell surface structure. Specifically, the amino acid residue positions in region B to be replaced may be selected from the group consisting of 34, 35, 39, 40, 41, 42, 44, 45 and 49.

Another embodiment of the present invention, provides a conjugate comprising a bacterial superantigen and an antibody moiety, wherein the superantigen is a low titer superantigen comprising regions A to E, which region A is a TCR binding site, and regions B to E determine the binding to MHC class II molecules; and the amino acid sequence of the superantigen is substituted so that no more than 15 amino acid residues in region C are replaced with different amino acids, such that the substituted superantigen has reduced seroreactivity compared to the superantigen from which it is derived; and wherein the antibody moiety is a full length antibody or any other molecule binding antibody active fragment, which is directed against a cancer-associated cell surface structure. In specific embodiments the cancer is selected from the group consisting of lung, breast, colon, kidney, pancreatic, ovarian, stomach, cervix and prostate cancer. The amino acid residue positions in region C to be replaced are selected from the group consisting of 74, 75, 78, 79, 81, 83 and 84.

Examples of superantigens include, but are not limited to staphylococcal enterotoxin (SE), a Streptococcus pyogenes exotoxin (SPE), a Staphylococcus aureus toxic shock-syndrome toxin (TSST-1), a streptococcal mitogenic exotoxin (SME) and a streptococcal superantigen (SSA). In specific embodiments, the staphylococcal enterotoxin is staphylococcal enterotoxin A (SEA) or staphylococcal enterotoxin E (SEE).

In specific embodiments, the conjugate may further comprise substitutions of no more than 15 amino acid residues in region A. The substitutions in region A may occur at the amino acid residue positions 20, 21, 24, 27, 173 or 204. Yet further, the conjugate may comprise substitutions of no more than 15 amino acid residues in region E. More particularly, the substitution of region E may occur at amino acid residue position 227.

In a further specific embodiment, the conjugate may comprise the SEE amino acid sequence including the substitutions of R20G, N21T, S24G, R27K, K79E, K81E, K83S, K84S and D227S or the SEE amino acid sequence including the substitutions of R20G, N21T, S24G, R27K, K79E, K81E, K83S, K84S and D227A. Yet further, the conjugate may comprise the amino acid sequence of SEQ ID NO: 2.

In further embodiments, the conjugate may comprise an antibody moiety, for example, but not limited to the Fab fragment. Specific Fab fragments may include C215Fab or 5T4Fab. Yet further, the conjugate may comprise the amino acid sequence of SEQ ID NO: 1.

Yet further, the conjugate may also comprise a cytokine, such as interleukin. In specific embodiments, the interleukin is IL2 or a derivative thereof having essentially the same biological activity of native IL2.

Another embodiment comprises a conjugate comprising a bacterial superantigen and an antibody moiety, wherein the superantigen is a low titer superantigen comprising regions A to E, which region A is a TCR binding site, and regions B to E determine the binding to MHC class II molecules; and the amino acid sequence of the superantigen is substituted so that no more than 15 amino acid residues in region D are replaced with different amino acids, such that the substituted superantigen has reduced seroreactivity compared to the superantigen from which it is derived; and wherein the antibody moiety is a full length antibody or any other molecule binding antibody active fragment, which is directed against a cancer-associated cell surface structure. The amino acid residue positions in region D to be replaced are selected from the group consisting of 187, 188, 189 and 190.

In another embodiment, it is provided a conjugate comprising a bacterial superantigen and an antibody moiety, wherein the superantigen is a low titer superantigen comprising regions A to E, which region A is a TCR binding site, and regions B to E determine the binding to MHC class II molecules; and the amino acid sequence of the superantigen is substituted so that no more than 15 amino acid residues in region E are replaced with different amino acids, such that the substituted superantigen has reduced seroreactivity compared to the superantigen from which it is derived; and wherein the antibody moiety is a full length antibody or any other molecule binding antibody active fragment, which is directed against a cancer-associated cell surface structure. In specific embodiments the staphylococcal enterotoxin is staphylococcal enterotoxin A (SEA) or staphylococcal enterotoxin E (SEE). Also, the amino acid residue positions in region E to be replaced are selected from the group consisting of 217, 220, 222, 223, 225 and 227.

In a specific embodiment, the conjugate further comprises substitutions of no more than 15 amino acid residues in region A. Specifically, the substitutions in region A may occur at the amino acid residue positions of 20, 21, 24, 27, 173 and 204.

In another specific embodiment, the conjugate further comprises substitutions of no more than 15 amino acid residues in region B in which the substitutions may occur at the amino acid residue positions of 34, 35, 39, 40, 41, 42, 44, 45 and 49.

Yet further, the conjugate may comprise substitutions of no more than 15 amino acid residues in region C. Specifically, the substitutions in region C occurs at the amino acid residue positions of 74, 75, 78, 79, 81, 83 and 84. Also, the conjugate may further comprise substitutions of no more than 15 amino acid residues in region D, in which the substitutions may occur at the amino acid residue positions of 187, 188, 189 and 190.

In other specific embodiment, it is provided a pharmaceutical composition comprising a therapeutically effective amount of a conjugate, wherein said conjugate comprises a bacterial superantigen and an antibody moiety, wherein the superantigen is a low titer superantigen comprising regions A to E, which region A is a TCR binding site, and regions B to E determine the binding to MHC class II molecules; and the amino acid sequence of the superantigen is substituted so that no more than 15 amino acid residues in region C are replaced with different amino acids, such that the substituted superantigen has reduced seroreactivity compared to the superantigen from which it is derived; and wherein the antibody moiety is a full length antibody or any other molecule binding antibody active fragment, which is directed against a cancer-associated cell surface structure. Specifically, the amino acid residue positions in region C to be replaced are selected from the group consisting of 74, 75, 78, 79, 81, 83 and 84.

In further embodiments, the pharmaceutical composition may comprise a conjugate comprising substitutions of no more than 15 amino acid residues in region A, in which the substitutions in region A occur at the amino acid residue positions of 20, 21, 24, 27, 173 and 204. Yet further, the pharmaceutical composition may also comprise substitutions of no more than 15 amino acid residues in region E. Specifically, the substitution of region E may be at amino acid residue position 227.

In specific embodiments, the pharmaceutical composition may comprise a conjugate comprising the SEE amino acid sequence (SEQ ID NO: 7) as well as the additional substitutions of R20G, N21T, S24G, R27K, K79E, K81E, K83S, K84S and D227S.

In another specific embodiment, the pharmaceutical composition may comprise the SEE amino acid sequence (SEQ ID NO: 7) as well as the additional substitutions of R20G, N21T, S24G, R27K, K79E, K81E, K83S, K84S and D227A. Yet further, the pharmaceutical composition comprises a conjugate that has the amino acid sequence of SEQ ID NO: 2.

In further specific embodiments, the pharmaceutical composition comprises an antibody moiety, for example a Fab fragment. Specifically, the Fab fragment is C215Fab or 5T4Fab. Yet further, the pharmaceutical composition comprises a conjugate that has the amino acid sequence of SEQ ID NO: 1. The pharmaceutical composition may further comprise a cytokine, such as an interleukin. The interleukin may be IL2 or a derivative thereof having essentially the same biological activity of native IL2.

Another embodiment of the present invention includes a method of treating cancer in a mammal by activation of the immune system of said mammal comprising administering to said mammal a therapeutically effective amount of a conjugate, wherein said conjugate comprises a bacterial superantigen and an antibody moiety, wherein the superantigen is a low titer superantigen comprising regions A to E, which region A is a TCR binding site, and regions B to E determine the binding to MHC class II molecules; and the amino acid sequence of the superantigen is substituted so that no more than 15 amino acid residues in region C are replaced with different amino acids, such that the substituted superantigen has reduced seroreactivity compared to the superantigen from which it is derived; and wherein the antibody moiety is a full length antibody or any other molecule binding antibody active fragment, which is directed against a cancer-associated cell surface structure. Examples of cancer include, but are not limited to lung, breast, colon, kidney, pancreatic, ovarian, stomach, cervix and prostate cancer. Specifically, the amino acid residue positions in region C to be replaced are selected from the group consisting of 74, 75, 78, 79, 81, 83 and 84.

In further embodiments, region A may also comprise substitutions of no more than 15 amino acid residues, in which the substitutions occur at the amino acid residue positions of 20, 21, 24, 27, 173 and 204. Also, region E may further comprise substitutions of no more than 15 amino acid residues. Specifically, a substitution of region E may be at amino acid residue position 227. The conjugate may comprise the SEE amino acid sequence (SEQ ID NO: 7) as well as the additional substitutions of R20G, N21T, S24G, R27K, K79E, K81E, K83S, K84S and D227S or the substitutions of R20G, N21T, S24G, R27K, K79E, K81E, K83S, K84S and D227A. Yet further, the conjugate has the amino acid sequence of SEQ ID NO: 2. Yet further, the conjugate may comprise an antibody moiety, for example, but not limited to the Fab fragment. Specific Fab fragments may include C215Fab or 5T4Fab. More particularly, the conjugate may comprise the amino acid sequence of SEQ ID NO: 1.
 

Claim 1 of 49 Claims

1. A conjugate comprising a bacterial superantigen and an antibody, wherein the superantigen is a variant of Staphylococcal enterotoxin E, reference SEQ ID NO: 7, and differs from Staphylococcal enterotoxin E in comprising amino acid substitutions as follows, wherein the positions of the amino acid substitutions are relative to the amino acid positions in reference SEQ ID NO: 7: (i) amino acid position 20 is glycine or a conserved variant thereof, amino acid position 21 is threonine or a conserved variant thereof, amino acid position 24 is glycine or a conserved variant thereof, amino acid position 27 is lysine or a conserved variant thereof, and amino acid position 227 is serine or alanine, or a conserved variant thereof; and (ii) wherein at least one amino acid in a region C is substituted with a different amino acid, such that the superantigen variant has reduced seroreactivity compared to the seroreactivity of Staphylococcal enterotoxin having the amino acid sequence of SEQ ID NO: 7, and the position of the amino acid substitution in region C is selected from the group consisting of amino acid positions 74, 75, 78, 79, 81, 83 and 84; and wherein the antibody binds 5T4 cancer antigen.

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